Adoptive cell therapy with young t cells

ABSTRACT

The invention provides a method of promoting regression of a cancer in a mammal comprising (i) culturing autologous T cells; (ii) expanding the cultured T cells; (iii) administering to the mammal nonmyeloablative lymphodepleting chemotherapy; and (iv) after administering nonmyeloablative lymphodepleting chemotherapy, administering to the mammal the expanded T cells, wherein the T cells administered to the mammal are about 19 to about 35 days old and have not been screened for specific tumor reactivity, whereupon the regression of the cancer in the mammal is promoted.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application claims the benefit of U.S. Provisional PatentApplication No. 61/237,889, filed Aug. 28, 2009, which is incorporatedby reference.

BACKGROUND OF THE INVENTION

Adoptive cell therapy (ACT) using tumor reactive T-cells following hostlymphodepletion can lead to positive, objective, and durable responsesin cancer patients. However, this therapy can involve sophisticated cellprocessing and in vitro lymphocyte culturing for extended periods. Theseprocedures have introduced technical, regulatory, and logisticchallenges to the successful use of antigen-specific T cells as abiological therapy. Accordingly, there is a need in the art for improvedmethods for treating cancer using adoptive cell therapy.

BRIEF SUMMARY OF THE INVENTION

An embodiment of the invention provides a method of promoting regressionof a cancer in a mammal comprising (i) culturing autologous T cells;(ii) expanding the cultured T cells using OKT3 antibody, IL-2, andfeeder lymphocytes, wherein the cultured T cells are enriched for CD8+ Tcells prior to expansion of the T cells; (iii) administering to themammal nonmyeloablative lymphodepleting chemotherapy; and (iv) afteradministering nonmyeloablative lymphodepleting chemotherapy,administering to the mammal the expanded T cells, wherein the T cellsadministered to the mammal are about 19 to about 35 days old and havenot been screened for specific tumor reactivity, whereupon theregression of the cancer in the mammal is promoted.

Another embodiment of the invention provides a method of promotingregression of a cancer in a mammal comprising (i) culturing autologous Tcells; (ii) expanding the cultured T cells using OKT3 antibody, IL-2,and feeder lymphocytes, wherein the cultured T cells are enriched forCD8+ T cells prior to expansion of the T cells; (iii) administering tothe mammal nonmyeloablative lymphodepleting chemotherapy; and (iv) afteradministering nonmyeloablative lymphodepleting chemotherapy,administering to the mammal the expanded T cells, wherein the T cellsadministered to the mammal are about 19 to about 29 days old, whereuponthe regression of the cancer in the mammal is promoted.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

FIG. 1A is a graph showing the frequency (percent) CD4⁺ markerexpression (y axis) per days in culture (x axis) for tumor infiltratinglymphocytes (TIL) generated from enzymatic digests of tumor (filledcircles) and TIL from tumor fragments (open squares) (p=0.02).

FIG. 1B is a graph showing the frequency (percent) CD8⁺ markerexpression (y axis) per days in culture (x axis) for TIL generated fromenzymatic digests of tumor (filled circles) and TIL from tumor fragments(open squares) (p=0.43).

FIG. 1C is a graph showing the frequency (percent) CD3-CD56⁺ markerexpression (y axis) per days in culture (x axis) for TIL generated fromenzymatic digests of tumor (filled circles) and TIL from tumor fragments(open squares) (p=0.02).

FIG. 2A is a graph showing the frequency (percent) CD27⁺ markerexpression of CD8⁺ cells (y axis) per days in culture (x axis) for TILgenerated from enzymatic digests of tumor (filled circles) and TIL fromtumor fragments (open squares) (p<0.0001).

FIG. 2B is a graph showing the frequency (percent) CD28⁺ markerexpression of CD8⁺ cells (y axis) per days in culture (x axis) for TILgenerated from enzymatic digests of tumor (filled circles) and TIL fromtumor fragments (open squares) (p=0.003).

FIG. 2C is a graph showing the frequency (percent) CD27⁺CD28⁺ markerexpression of CD8⁺ cells (y axis) per days in culture (x axis) for TILgenerated from enzymatic digests of tumor (filled circles) and TIL fromtumor fragments (open squares) (p<0.00001).

FIG. 3 is a graph showing mean telomere length (kb) (y axis) for TILcultured per days in culture (x axis) (p<0.001).

FIG. 4A is a computed tomography (CT) scan showing metastatic melanomalesions (arrows) in a first patient before CD8+ enriched young TILtherapy in the sacrum and ilium (top left), lung (middle left) andspleen (lower left) and 2 months after treatment in the sacrum and ilium(top right), lung (middle right) and spleen (lower right).

FIG. 4B (left panel) is a photograph showing subcutaneous melanomaaround the ear and in the auditory canal in a second patient nine daysprior to CD8+ enriched young TIL therapy; middle panel is a photographshowing gross necrosis of the melanoma 11 days following treatment;right panel is a photograph showing a partial response 76 days aftertreatment.

FIG. 4C (left panel) is a CT scan showing (from top to bottom)mediastinal, lung, nodal and subcutaneous metastatic deposits (arrows)in a third patient before (left) and one month after (right) treatmentwith CD8+ enriched young TIL.

FIG. 5A is a graph showing the percentage of lymphocytes in initialsuspension (y axis) for TIL that grew to use for treatment (>5×10⁷ cellsin 28 days, Rx TIL) or TIL for which growth was insufficient fortreatment (no growth) (x axis). Black bars indicate median values of thepopulations. p2=5×10⁻⁸.

FIG. 5B is a graph showing the number of days in culture (y axis) forTIL in prior protocols in which the TIL underwent individualized testingfor tumor reactivity (Specific TIL, n=92) or CD8+ enriched young TIL(CD8+ Young TIL, n=55) (x axis) for non-responding (NR) patients (whitebars) or objective responders (OR) (black bars). Standard error bars areshown. p2=0.04. NS=not significant.

FIG. 6A is a graph showing interferon (IFN)-gamma secretion (pg/ml) (yaxis) of CD8+ enriched young TIL administered to patients (x axis) afternon-myeloablative chemotherapy (NMA) upon incubation with autologous(black bars), HLA-matched (hatched bars), or HLA-mismatched (white bars)tumors. Patients with specific tumor recognition (greater than 200 pg/mlIFN-gamma and 2×HLA-mis-matched tumor) are boxed. Offscale >5000 pg/ml.

FIG. 6B is a graph showing interferon (IFN)-gamma secretion (pg/ml) (yaxis) of CD8+ enriched young TIL administered to patients (x axis) after6Gy total body irradiation (TBI) upon incubation with autologous (blackbars), HLA-matched (hatched bars), or HLA-mismatched (white bars)tumors. Patients with specific tumor recognition (greater than 200 pg/mlIFN-gamma and 2×HLA-mis-matched tumor) are boxed. Offscale >5000 pg/ml.

FIG. 7A is a graph showing average absolute lymphocyte cell (ALC) count(cells/microliter) (y axis) for all patients who received CD8+ enrichedyoung TIL with NMA (n=33) (black squares) or all patients who receivedextensively expanded, tumor selected TIL with NMA as their firsttreatment (n=33) (historic control) (white squares) over time (days)relative to TIL infusion (x axis). Arrows: IL-2 therapy. Vertical bars:standard error.

FIG. 7B is a graph showing average absolute lymphocyte cell (ALC) count(cells/microliter) (y axis) for all patients who received CD8+ enrichedyoung TIL with 6Gy TBI (n=23) (black triangles) or all patients whoreceived extensively expanded, tumor selected TIL with 12Gy TBI (n=25)(historic control) (white triangles) over time (days) relative to TILinfusion (x axis). Arrows: IL-2 therapy. Vertical bars: standard error.

FIG. 7C is a graph showing CD8+ absolute lymphocyte count (ALC) (cellsper microliter) (y axis) for each non-responding (NR) patient orobjective responders (OR) (x axis). Black bars: mean ALC for thepopulation. p=0.002.

FIG. 7D is a graph showing CD4+ absolute lymphocyte count (ALC) (cellsper microliter) (y axis) for each non-responding (NR) patient orobjective responders (OR) (x axis). Black bars: mean ALC for thepopulation. p=0.5.

DETAILED DESCRIPTION OF THE INVENTION

An embodiment of the present invention provides a method of promotingthe regression of a cancer in a mammal. The method comprises (i)culturing autologous T cells; (ii) expanding the cultured T cells usingOKT3 antibody, IL-2, and feeder lymphocytes, wherein the cultured Tcells are enriched for CD8+ T cells prior to expansion of the T cells;(iii) administering to the mammal nonmyeloablative lymphodepletingchemotherapy; and (iv) after administering nonmyeloablativelymphodepleting chemotherapy, administering to the mammal the expanded Tcells, wherein the T cells administered to the mammal are about 19 toabout 35 days old and have not been screened for specific tumorreactivity, whereupon the regression of the cancer in the mammal ispromoted. In some embodiments, the administered T cells are less thanabout 35 days old, e.g., about 19 to about 26 days old.

The inventive methods provide numerous advantages. For example, T cellsthat are about 19 to about 35 days old are believed to provide improvedin vivo proliferation, survival, and antitumor activity as compared to Tcells that are about 44 days old or older. In addition, because theinventive methods include nonmyeloablative chemotherapy, the inventivemethods can advantageously be used to treat patients that would not beeligible for treatments that involve total body irradiation (TBI) suchas, for example, patients that have already undergone myeloablativetherapy, e.g., radiotherapy, prior to treatment; patients with comorbidconditions; and patients with less than 2×10⁶ CD34⁺ cells/kg. Moreover,the period of time required to generate T cells for adoptive celltherapy (ACT) may be shortened from an average of about 44 days to arange of about 19 to about 35 days (or less than about 35 days, e.g.,about 19 to about 29 days, or about 19 to about 26 days). Accordingly,more patients may be treated before their disease burden progresses to astage in which administration of ACT may no longer be safe or possible.Furthermore, because preferred embodiments of the inventive methods donot require in vitro testing of specific antigen reactivity prior toadministration, the inventive methods reduce the time, expense, andlabor associated with the treatment of patients. Additionally, theinventive methods may advantageously administer T cells that are pooledfrom bulk cultures instead of those derived from microcultures. Thedevelopment of a simpler and faster method to generate clinicallyeffective T cells is believed to aid in the more widespread use ofadoptive cell therapy. The inventive methods also advantageously utilizeT cell cultures that could be falsely predicted to be unreactive in vivoby in vitro testing of specific antigen reactivity. Because T cellcultures generated from a single tumor specimen have diverse specificreactivities, the lack of in vitro antigen reactivity testingadvantageously avoids having to choose only a few T cell cultures toexpand, and therefore provides a more diverse repertoire of tumorreactivities to be administered to the patient. T cells that are about19 to about 35 days old also contain a greater diversity of cells and ahigher frequency of CD4⁺ cells than T cells that are about 44 days old.In addition, one or more aspects (e.g., but not limited to, culturingand/or expanding) of the inventive methods may be automatable.

An embodiment of the method comprises culturing autologous T cells.Tumor samples are obtained from patients and a single cell suspension isobtained. The single cell suspension can be obtained in any suitablemanner, e.g., mechanically (disaggregating the tumor using, e.g., agentleMACS™ Dissociator, Miltenyi Biotec, Auburn, Calif.) orenzymatically (e.g., collagenase or DNase). Single-cell suspensions oftumor enzymatic digests are cultured in interleukin-2 (IL-2), e.g., inmultiple wells. The cells are cultured until confluence (e.g., about2×10⁶ lymphocytes), e.g., from about 5 to about 21 days, preferably fromabout 10 to about 14 days. For example, the cells may be cultured from 5days, 5.5 days, or 5.8 days to 21 days, 21.5 days, or 21.8 days,preferably from 10 days, 10.5 days, or 10.8 days to 14 days, 14.5 days,or 14.8 days.

An embodiment of the method comprises expanding cultured T cells. Thecultured T cells are pooled and rapidly expanded. Rapid expansionprovides an increase in the number of antigen-specific T-cells of atleast about 50-fold (e.g., 50-, 60-, 70-, 80-, 90-, or 100-fold, orgreater) over a period of about 10 to about 14 days, preferably about 14days. More preferably, rapid expansion provides an increase of at leastabout 200-fold (e.g., 200-, 300-, 400-, 500-, 600-, 700-, 800-, 900-, orgreater) over a period of about 10 to about 14 days, preferably about 14days. Most preferably, rapid expansion provides an increase of at leastabout 1000-fold over a period of about 10 to about 14 days, preferablyabout 14 days. Preferably, rapid expansion provides an increase of about1000-fold over a period of about 14 days.

Expansion can be accomplished by any of a number of methods as are knownin the art. For example, T cells can be rapidly expanded usingnon-specific T-cell receptor stimulation in the presence of feederlymphocytes and either interleukin-2 (IL-2) or interleukin-15 (IL-15),with IL-2 being preferred. The non-specific T-cell receptor stimulus caninclude around 30 ng/ml of OKT3, a mouse monoclonal anti-CD3 antibody(available from Ortho-McNeil®, Raritan, N.J.). Alternatively, T cellscan be rapidly expanded by stimulation of peripheral blood mononuclearcells (PBMC) in vitro with one or more antigens (including antigenicportions thereof, such as epitope(s), or a cell) of the cancer, whichcan be optionally expressed from a vector, such as an human leukocyteantigen A2 (HLA-A2) binding peptide, e.g., 0.3 μM MART-1:26-35 (27 L) orgp100:209-217 (210M), in the presence of a T-cell growth factor, such as300 IU/ml IL-2 or IL-15, with IL-2 being preferred. The in vitro-inducedT-cells are rapidly expanded by re-stimulation with the same antigen(s)of the cancer pulsed onto HLA-A2-expressing antigen-presenting cells.Alternatively, the T-cells can be re-stimulated with irradiated,autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytesand IL-2, for example.

An embodiment of the method comprises administering to the mammalnonmyeloablative lymphodepleting chemotherapy. The nonmyeloablativelymphodepleting chemotherapy can be any suitable such therapy, which canbe administered by any suitable route. The nonmyeloablativelymphodepleting chemotherapy can comprise, for example, theadministration of cyclophosphamide and fludarabine, particularly if thecancer is melanoma, which can be metastatic. A preferred route ofadministering cyclophosphamide and fludarabine is intravenously.Likewise, any suitable dose of cyclophosphamide and fludarabine can beadministered. Preferably, around 60 mg/kg of cyclophosphamide isadministered for two days after which around 25 mg/m² fludarabine isadministered for five days, particularly if the cancer is melanoma.

An embodiment of the method comprises, after administering thenonmyeloablative lymphodepleting chemotherapy, administering to themammal the expanded T cells, wherein the T cells administered to themammal are about 19 to about 35 days old. For example, the administeredcells may be 19, 19.5, or 19.8 to 35, 35.5, or 35.8 days old. In someembodiments, the T cells administered to the mammal are about 19 toabout 29 or about 23 to about 29 days old, or about 26 days old. Forexample, the administered cells may be 19, 19.5, or 19.8 to 29, 29.5, or29.8 days old; 23, 23.5, or 23.8 to 29, 29.5, or 29.8 days old; or 26,26.5, or 26.8 days old. In this regard, the T cells that areadministered to the mammal according to an embodiment of the inventionare “young” T cells, i.e., minimally cultured T cells.

Young T cell cultures that are administered to the mammal in accordancewith an embodiment of the invention advantageously have featuresassociated with in vivo persistence, proliferation, and antitumoractivity. For example, young T cell cultures have a higher expression ofCD27 and/or CD28 than T cells that are about 44 days old. Without beingbound to a particular theory, it is believed that CD27 and CD28 areassociated with proliferation, in vivo persistence, and a lessdifferentiated state of T cells (the increased differentiation of Tcells is believed to negatively affect the capacity of T cells tofunction in vivo). T cells expressing higher levels of CD27 are believedto have better antitumor activity than CD27-low cells. Moreover, young Tcell cultures have a higher frequency of CD4⁺ cells than T cells thatare about 44 days old.

In addition, young T cell cultures have a mean telomere length that islonger than that of T cells that are about 44 days old. Without beingbound to a particular theory, it is believed that T cells lose anestimated telomere length of 0.8 kb per week in culture, and that youngT cell cultures have telomeres that are about 1.4 kb longer than T cellsthat are about 44 days old. Without being bound to a particular theory,it is believed that longer telomere lengths are associated with positiveobjective clinical responses in patients and persistence of the cells invivo.

The T-cells can be administered by any suitable route as known in theart. Preferably, the T-cells are administered as an intra-arterial orintravenous infusion, which preferably lasts about 30 to about 60minutes. Other examples of routes of administration includeintraperitoneal, intrathecal and intralymphatic.

Likewise, any suitable dose of T-cells can be administered. Preferably,from about 1.0×10¹⁰ T-cells to about 13.7×10¹⁰ T-cells are administered,with an average of around 5.0×10¹⁰ T-cells, particularly if the canceris melanoma. Alternatively, from about 1.2×10¹⁰ to about 4.3×10¹⁰T-cells are administered.

In a preferred embodiment, the T cells are not tested for specific tumorreactivity to identify tumor reactive T cells prior to administration tothe patient. Specific tumor reactivity can be tested by any method knownin the art, e.g., by measuring cytokine release (e.g., interferon-γ)following co-culture with tumor cells. The inventive methodsadvantageously make it possible to promote regression of cancer in amammal by administering T cells to the mammal without the necessity ofprior screening for specific tumor recognition. Embodiments of themethods may, if desired, test the T cells for potency in anon-antigen-specific manner prior to administering the T cells to themammal. T cell potency may be tested, e.g., by a non-specific potencyassay measuring cytokine release following OKT3 stimulation. T cells maybe considered potent if, for example, interferon (IFN) release isgreater than about 50 pg/mL, greater than about 100 pg/mL, greater thanabout 150 pg/mL, or greater than about 200 pg/mL. A less desiredembodiment of the method comprises testing the expanded T cells forspecific tumor reactivity to identify tumor-reactive T cells.

Another embodiment of the invention provides a method of promotingregression of a cancer in a mammal comprising (i) culturing autologous Tcells; (ii) expanding the cultured T cells using OKT3 antibody, IL-2,and feeder lymphocytes, wherein the cultured T cells are enriched forCD8+ T cells prior to expansion of the T cells; (iii) administering tothe mammal nonmyeloablative lymphodepleting chemotherapy; and (iv) afteradministering nonmyeloablative lymphodepleting chemotherapy,administering to the mammal the expanded T cells, wherein the T cellsadministered to the mammal are about 19 to about 29 days old, whereuponthe regression of the cancer in the mammal is promoted. For example, theT cells administered to the mammal may be 19, 19.5, 19.8 to 29, 29.5, or29.8 days old. An embodiment of the method comprises culturingautologous T cells as described herein from about 5 days to about 15days. For example, the T cells may be cultured from 5, 5.5, or 5.8 daysto 15, 15.5, or 15.8 days. The method further comprises expanding thecultured T cells and administering to the mammal nonmyeloablativelymphodepleting chemotherapy as described herein. After administeringnonmyeloablative lymphodepleting chemotherapy, the method comprisesadministering to the mammal the expanded T cells as described herein,whereupon the regression of the cancer in the mammal is promoted. In apreferred embodiment of the method, the administered T cells have notbeen screened for specific tumor reactivity.

An embodiment of the method comprises enriching cultured T cells forCD8⁺ T cells prior to rapid expansion of the cells. Following culture ofthe T cells in IL-2, the T cells are depleted of CD4⁺ cells and enrichedfor CD8⁺ cells using, for example, a CD8 microbead separation (e.g.,using a CliniMACS^(plus) CD8 microbead system (Miltenyi Biotec)).Without being bound to a particular theory, it is believed that CD4⁺,CD25⁺ regulatory T-cells can impede anti-tumor responses. Accordingly,it is believed that enriching cultured T cells for CD8⁺ T cells andreducing or eliminating CD4⁺ cells may improve the impact of adoptivelytransferred anti-tumor CD8⁺ cells, improve the response rates inpatients, and/or reduce the toxicities seen by production of cytokinesby CD4⁺ cells. Moreover, it is believed that CD8⁺ enrichment of some Tcell cultures reveals in vitro tumor recognition that may not be evidentin the bulk culture, and improved in vitro recognition of tumor in othercultures. Additionally, the enriched CD8⁺ young T cells are believed tobehave more reliably and predictably in clinical scale rapid expansionsthan the bulk T cells.

In an embodiment of the method, a T-cell growth factor that promotes thegrowth and activation of the autologous T cells is administered to themammal either concomitantly with the autologous T cells or subsequentlyto the autologous T cells. The T-cell growth factor can be any suitablegrowth factor that promotes the growth and activation of the autologousT-cells. Examples of suitable T-cell growth factors include interleukin(IL)-2, IL-7, IL-15, and IL-12, which can be used alone or in variouscombinations, such as IL-2 and IL-7, IL-2 and IL-15, IL-7 and IL-15,IL-2, IL-7 and IL-15, IL-12 and IL-7, IL-12 and IL-15, or IL-12 and IL2.IL-12 is a preferred T-cell growth factor.

In an embodiment of the method, the autologous T-cells are modified toexpress a T-cell growth factor that promotes the growth and activationof the autologous T-cells. Suitable T-cell growth factors include, forexample, any of those described above. Suitable methods of modificationare known in the art. See, for instance, Sambrook et al., MolecularCloning: A Laboratory Manual, 3^(rd) ed., Cold Spring Harbor Press, ColdSpring Harbor, N.Y. 2001; and Ausubel et al., Current Protocols inMolecular Biology, Greene Publishing Associates and John Wiley & Sons,NY, 1994. Desirably, modified autologous T-cells express the T-cellgrowth factor at high levels. T-cell growth factor coding sequences,such as that of IL-12, are readily available in the art, as arepromoters, the operable linkage of which to a T-cell growth factorcoding sequence promote high-level expression.

In some embodiments, it is believed, two cytokines are more effectivethan a single cytokine, and three cytokines, e.g., IL-2, IL-7 and IL-15,are better than any two cytokines. It is believed that IL-15 enhances atumor-specific CD8⁺ T-cell response. In this regard, the administrationof IL-15-cultured cells with IL-2 (such as a bolus injection) can beparticularly efficacious.

The T-cell growth factor can be administered by any suitable route. Ifmore than one T-cell growth factor is administered, they can beadministered simultaneously or sequentially, in any order, and by thesame route or different routes. Preferably, the T-cell growth factor,such as IL-2, is administered intravenously as a bolus injection.Desirably, the dosage of the T-cell growth factor, such as IL-2, is whatis considered by those of ordinary skill in the art to be high.Preferably, a dose of about 720,000 IU/kg of IL-2 is administered threetimes daily until tolerance, particularly when the cancer is melanoma.Preferably, about 5 to about 15 doses of IL-2 are administered, with anaverage of around 9 doses.

T-cells can recognize any of the unique antigens produced as a result ofthe estimated 10,000 genetic mutations encoded by each tumor cellgenome. The antigen, however, need not be unique. T-cells can recognizeone or more antigens of a cancer, including an antigenic portion of oneor more antigens, such as an epitope, or a cell of the cancer. An“antigen of a cancer” and an “antigen of the cancer” are intended toencompass all of the aforementioned antigens. If the cancer is melanoma,such as metastatic melanoma, preferably the T-cells recognize MART-1(such as MART-1:26-35 (27 L)), gp100 (such as gp100:209-217 (210M)), ora “unique” or patient-specific antigen derived from a tumor-encodedmutation. Other suitable melanoma antigens which may be recognized byT-cells can include, but are not limited to, NY-ESO-1, tyrosinase tumorantigen, tyrosinase related protein (TRP)-1, TRP-2, VEGFR-2, and amember of the MAGE family of proteins, e.g., MAGE-A1, MAGE A2, MAGE-A3,MAGE-A6, and MAGE 12. T cells can also recognize antigens such as, forexample, telomerase, p53, HER2/neu, mesothelin, carcinoembryonicantigen, or prostate-specific antigen, for treatment of lung carcinoma,breast cancer, colon cancer, prostate cancer, and the like.

In an embodiment of the method, the autologous T-cells are modified toexpress a T cell receptor (TCR) having antigenic specificity for acancer antigen, e.g., any of the cancer antigens described herein.Suitable TCRs include, for example, those with antigenic specificity fora melanoma antigen, e.g., gp100 or MART-1. Suitable methods ofmodification are known in the art. See, for instance, Sambrook andAusubel, supra. For example, the T cells may be transduced to express aT cell receptor (TCR) having antigenic specificity for a cancer antigenusing transduction techniques described in Heemskerk et al. Hum GeneTher. 19:496-510 (2008) and Johnson et al. Blood 114:535-46 (2009).

With respect to the inventive methods, the cancer can be any cancer,including any of acute lymphocytic cancer, acute myeloid leukemia,alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer,cancer of the anus, anal canal, or anorectum, cancer of the eye, cancerof the intrahepatic bile duct, cancer of the joints, cancer of the neck,gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear,cancer of the vulva, chronic lymphocytic leukemia, chronic myeloidcancer, cervical cancer, glioma, Hodgkin lymphoma, hypopharynx cancer,kidney cancer, larynx cancer, liver cancer, lung cancer, malignantmesothelioma, melanoma, multiple myeloma, nasopharynx cancer,non-Hodgkin lymphoma, ovarian cancer, peritoneum, omentum, and mesenterycancer, pharynx cancer, prostate cancer, rectal cancer, renal cancer,skin cancer, soft tissue cancer, testicular cancer, thyroid cancer,ureter cancer, urinary bladder cancer, and digestive tract cancer suchas, e.g., esophageal cancer, gastric cancer, pancreatic cancer, stomachcancer, small intestine cancer, gastrointestinal carcinoid tumor, cancerof the oral cavity, colon cancer, and hepatobiliary cancer. A preferredcancer is melanoma. A particularly preferred cancer is metastaticmelanoma.

As used herein, the term “mammal” refers to any mammal, including, butnot limited to, mammals of the order Rodentia, such as mice andhamsters, and mammals of the order Logomorpha, such as rabbits. It ispreferred that the mammals are from the order Carnivora, includingFelines (cats) and Canines (dogs). It is more preferred that the mammalsare from the order Artiodactyla, including Bovines (cows) and Swines(pigs) or of the order Perssodactyla, including Equines (horses). It ismost preferred that the mammals are of the order Primates, Ceboids, orSimoids (monkeys) or of the order Anthropoids (humans and apes). Anespecially preferred mammal is the human.

The term “regression,” as well as words stemming therefrom, as usedherein, does not necessarily imply 100% or complete regression. Rather,there are varying degrees of regression of which one of ordinary skillin the art recognizes as having a potential benefit or therapeuticeffect. In this respect, the inventive methods can provide any amount ofany level of regression of cancer in a mammal. Furthermore, theregression provided by the inventive method can include regression ofone or more conditions or symptoms of the disease, e.g., cancer. Also,for purposes herein, “regression” can encompass delaying the onset ofthe disease, or a symptom or condition thereof.

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

Comparative Example 1

This example demonstrates the generation of “standard”tumor-infiltrating lymphocytes (TIL).

“Standard” TIL were generated as generally described in Example 1 (setout after the Comparative Examples below), except that a standard TILculture would be cultured to generate 5×10⁷ lymphocytes from eachoriginal well of tumor fragment or digest after 21-36 days. Standard TILwere propagated by splitting an individual confluent well into twodaughter wells, and maintaining each initial fragment or well of digestas an independent culture. Each standard TIL culture was split multipletimes until it comprised confluent growth of 24 daughter wells generatedfrom one original 2-ml well. TIL were assayed for activity when theculture generated 5×10⁷ lymphocytes from each original well of tumorfragment or digest (after 21-36 days).

Specific reactivity of TIL was assessed by interferon-γ (IFN-γ) releaseassay. TIL were washed prior to use to remove IL-2, then culturedovernight with autologous, HLA-matched, or HLA-mismatched tumor cells ata ratio of 1:1. Single cell suspensions of fresh tumor digests wereprepared as targets from autologous or allogeneic melanoma specimens byovernight digestion of macerated tumor fragments in media containingcollagenase, hyaluronidase, and DNAse. The single cell suspension waswashed twice with HBSS and aliquots were cryopreserved. Targets werethawed on the day of coculture, and viable tumor cells were assessed bytrypan blue exclusion. The supernatant from each coculture was thenassayed for IFN-γ by ELISA (Pierce/Endogen) according to themanufacturer recommendations. A TIL culture was defined as possessingspecific reactivity if IFN-γ release was twice background (coculture ofTIL with HLA-mismatched tumors) and at least 200 pg/mL unless otherwisenoted.

Rapid expansion was performed as described in Example 1.

This example demonstrated the generation of “standard”tumor-infiltrating lymphocytes (TIL).

Comparative Example 2

This example demonstrates that tests for in vitro reactivity mayunderestimate the number of tumor-reactive TIL cultures.

When multiple independent TIL cultures are generated from a singlemelanoma tumor and screened for tumor recognition they often exhibitmultiple patterns of reactivity. A representative example from anHLA-A2+ patient is shown in Table 1. In this example, six independentTIL cultures were generated from tumor fragments (F1-F6) and fourindependent TIL cultures were generated from enzymatic single celldigests (D1-D4) of one melanoma tumor as described in ComparativeExample 1. All cultures were the same age and were evaluated in the samecoculture assay. When stimulated with HLA-A2+ and HLA-negative melanomalines, five of the ten independent TIL cultures (50%) had specificreactivity by objective criteria (IFNγ release that was greater than 200pg/ml and more than twice the highest HLA-mismatched control). Noautologous tumor cell line was available from this patient, but when theTIL cultures were stimulated with uncultured autologous tumor cells andHLA-mismatched tumor cell controls, seven of ten independent TILcultures (70%) had specific reactivity. Some TIL cultures recognized onesource of tumor antigen but not the other source, suggesting thepresence of multiple antigen reactive lymphocyte populations in theseTIL cultures.

TABLE 1 Melanoma Cell Line Fresh Tumor TIL* 888 938 526 624 2515 2547Auto- A2+ A2− A2− A2+ A2+ Reactivity† A2− A2- logous Reactivity IFNg(pg/ml) IFNg (pg/ml) F1 49 63 73 120 − 73 193 377 − F2 83 69 1470 1676 +63 79 360 + F3 40 62 71 95 − 55 45 268 + F4 51 44 122 230 + 81 101 206 +F5 35 35 84 248 + 48 56 94 − F6 13 16 19 22 − 16 100 162 − D1 66 44 394395 + 175 488 3340 + D2 77 52 140 140 − 257 552 5380 + D3 280 224 15611420 + 528 1394 4870 + D4 168 145 200 269 − 350 902 3110 + Totalpositive (%) 5 (50) 7 (70) *Results for ten independent TIL culturesderived from a single tumor from an HLA-A2+ patient are shown. Sixcultures prefixed by an “F” were derived from tumor fragments and fourcultures prefixed by a “D” were derived from enzymatically-prepareddigests. †Bold numbers indicate a positive test defined as at least 200pg/mL and greater than twice IFN-γ released by coculture withHLA-mismatched controls. The TIL culture was considered reactive ifeither HLA-matched target was positive.

Some TIL cultures do not specifically recognize HLA-matched melanomacell lines, but do recognize autologous tumor cells (such as F3, D2, andD4 in Table 1).

This example demonstrated that tests for in vitro reactivity mayunderestimate the number of tumor-reactive TIL cultures.

Comparative Example 3

This example demonstrates that tests for in vitro reactivity mayunderestimate the number of tumor-reactive TIL cultures.

The initial results from a cytokine release assay for several TILcultures from a patient with a large, inoperable scalp lesion andmultiple lung metastases showed no reactivity against HLA-matched tumorlines as targets. In this assay no autologous tumor cells wereavailable. Since no reactive TIL were available, the patient wasdischarged from the protocol and the TIL cultures were cryopreserved.After several additional weeks, an autologous tumor cell line wasestablished. When the TIL were thawed and the autologous tumor was usedas a target, it revealed significant tumor specific recognition byseveral cultures, including TIL F7. The patient was contacted andconsented to receive the F7 TIL cells with high dose IL-2 following anon-myeloablative lymphodepleting preparative chemotherapy on theexperimental clinical protocol (Dudley et al. J. Clin. Oncol. 23:2346-57(2005)). The patient experienced a dramatic regression of tumor at allsites and has an ongoing objective response now more than three yearsafter initial TIL treatment. Without the appropriate autologous tumortarget cells, the F7 TIL culture could not have been used for treatmentin this protocol, which requires evidence of tumor recognition as partof the certificate of release for the cell product.

This example demonstrated that tests for in vitro reactivity mayunderestimate the number of tumor-reactive TIL cultures.

Comparative Example 4

This example demonstrates that tests for in vitro reactivity mayunderestimate the number of tumor-reactive TIL cultures.

To quantify the potential clinical impact of autologous tumor targetavailability, we undertook a retrospective analysis of sequentialmelanoma samples (Table 2).

TABLE 2 Patients Tumors (% of total) (% of total) Tissue received inCell Processing Facility  83 (100%) 142 (100%) One or more independentTIL culture growth 72 (87%) 116 (82%)  to >10 million cells¹ Sharedmelanoma Ag recognition (HLA 30 (36%) 40 (26%) matched tumor cell linerecognition²) Unique Ag recognition (autologous 11 (13%) 21 (15%)cryopreserved tumor recognition) Total TIL available for treatment 41(49%) 61 (43%) ¹Growth positive TIL was considered 10 million cells fromany single independent culture. Typically 20 to 50 million cells wereobtained in 3 to 4 weeks. ²Tumor recognition was defined by IFNγ releaseassay with specific release being greater than 200 pg/ml and twicebackground.

During this ten month period, 142 tumors were processed from 83patients. TIL failed to grow from 26 tumors, resulting in 11 patientswho had no TIL to screen for tumor reactivity. TIL cultures weresuccessfully screened by coculture assay and IFNγ ELISA from one or moreindependent TIL culture from the remaining 116 tumors from 72 patients,including 49 HLA-A2 patients. Forty tumors (26%) from 30 differentpatients (36%) exhibited specific recognition of HLA-matched melanomatumor lines, including 24 HLA-A2+ patients and six HLA-A2 negativepatients. Additional specific tumor recognition was revealed for 21tumors (15%) from 11 patients (13%) by using autologous tumor cells asstimulator cells in cytokine release assays. These data emphasize thattests for in vitro reactivity may underestimate the number oftumor-reactive TIL cultures unless ideal tumor target cells areavailable. Based in part on these results, we initiated an investigationof alternative strategies for the production of TIL cultures for use inACT trials, including the use of early TIL cultures with untestedantigen reactivities.

This example demonstrated that tests for in vitro reactivity mayunderestimate the number of tumor-reactive TIL cultures.

Example 1

This example demonstrates the generation of “young” tumor-infiltratinglymphocytes (TIL).

Patients were entered into clinical protocols and signed informedconsents that were approved by the Institutional Review Board of theNational Cancer Institute prior to tumor resection. TIL were prepared aspreviously described in detail (Dudley et al. J. Immunother. 26:332-42(2003)). Briefly, multiple independent TIL cultures were set up usingenzymatic digests and tumor fragments (1 mm³) procured by sharpdissection. TIL from tumor digests were generated by culturingsingle-cell suspensions (5×10⁵/ml) obtained by overnight enzymaticdigestion of tumor fragments in media containing collagenase,hyaluronidase, and DNAse. Fragments and digests were initiated in 2 mlwells of complete medium (CM) and IL2 (6000 IU/ml, Chiron Corp.,Emeryville, Calif.) in a humidified 37° C. incubator with 5% CO₂. CMconsisted of RPMI1640 with glutamine, plus 10% human AB serum, 25 mMHEPES, 10 μg/ml gentamicin, and 5.5×10⁻⁵M 2-mercaptoethanol. Five daysafter initiation, one half of the media was aspirated from the wells andreplaced with fresh CM and IL-2, and media was replaced every two tothree days thereafter as needed. Under these conditions, lymphocyteswill first lyse the adherent cells in the well, and then begin tomultiply and grow.

Young TIL were defined as cultures which had just expanded to confluentgrowth of the original 2-ml well and eliminated adherent tumor cells,typically about 10-18 days after initiation. In practice, this was about2×10⁶ lymphocytes from each original tumor fragment or digest well. Bypooling all the wells in a single 24 well plate, approximately 5×10⁷young TIL cells would be obtained.

Rapid expansions of the culture was performed using the Rapid ExpansionProtocol (REP) as previously described (Dudley et al. J. Immunother.26:332-42 (2003) and Riddell et al. J. Immunol. Methods 128:189-201(1990)). Briefly, TIL cells were cultured in T25 flasks with a 200 foldexcess of irradiated (40 Gy) allogeneic peripheral blood mononuclear“feeder” cells in complete medium (CM) with 30 ng/ml anti-CD3 antibodyand 6000 IU/ml IL-2. Half of the media was exchanged on day 5 using CMwith 6000 IU/ml IL-2, and cells were split as needed thereafter. Cellactivity was assessed on day 14 of the rapid expansion (TIL expanded anaverage of more than 3000 fold).

This example demonstrated the generation of “young” tumor-infiltratinglymphocytes (TIL) having an age of 24-32 days.

Example 2

This example demonstrates the testing of young TIL cultures for specificreactivity.

When cultures designated for young TIL generation expanded to confluencein 2-ml wells, they were tested for specific reactivity. Because theyoung TIL were set up in large numbers (typically groups of 24 pertumor) it was not feasible to count each TIL culture individually. Theyoung TIL specificity assay measures activity per volume rather thanactivity per cell. Each well was mixed thoroughly, and exactly onehundred microliters of lymphocytes (estimated 1×10⁵ cells) was washedand cocultured with 1×10⁵ autologous or HLA-mismatched tumor cellsovernight. IFN-γ release was then measured with enzyme-linkedimmunosorbent assay (ELISA). A TIL culture was defined as possessingspecific reactivity if IFN-γ release was twice background (coculture ofTIL with HLA-mismatched tumors) and at least 200 pg/mL unless otherwisenoted.

This example demonstrated the testing of young TIL cultures for specificreactivity.

Example 3

This example demonstrates the generation of young lymphocytes foradoptive transfer therapy by growth of TIL from melanoma tumors.

The materials used in this Example include: Ca⁺⁺-, Mg⁺⁺-, Phenolred-free Hanks' balanced salt solution (HBSS) (BioWhittaker); RPMI 1640with L-Glutamine (Bio Whittaker; Walkersville, Md.); AIM-V medium,(GIBCO, Life Technologies; Grand Island, N.Y.); Human serum, type AB(Approved source with appropriate COA); Recombinant human IL-2 (10⁶CU/ml, Chiron Corp., Emeryville, Calif.) (Note: 1000 Cetus Units(CU)=6000 International Units (IU)); Collagenase, type IV, 1 g/literstock enzyme medium (Sigma-Aldrich; St. Louis, Mo.); Hyaluronidase, typeV, 100 mg/liter stock enzyme medium (Sigma-Aldrich); Deoxyribonuclease(DNAase), type IV, 30,000 units/liter stock enzyme medium(Sigma-Aldrich); Gentamicin sulfate, 10 mg/ml, stock (BioWhittaker)(Omit if patient is allergic to gentamicin); L-Glutamine, 29.2 mg/ml,stock (Mediatech; Herndon, Va.); Penicillin/Streptomycin (10,000 unitsPen/ml, 10,000 μg Strep/ml) (BioWhittaker) (Omit if patient is allergicto penicillin/streptomycin); OKT3 (Ortho-anti-CD3) (Orthoclone);Fungizone, 250 μg/ml, stock (Bristol-Myers Squibb Co.; Princeton, N.J.)(Omit if patient is allergic to fungizone); Ciprofloxacin, 10 mg/mlstock, (Bayer, West Haven, Conn.) (Omit if patient is allergic tociprofloxacin); Lymphocyte separation medium (LSM), (ICN Biomed, Inc;Avrora, Ohio); Albumin (Human) 25%, USP, (100 ml, Baxter Healthcare Co,;Glendale, Calif.); Nalge filters; 0.8, 0.45, and 0.22 μm (1 package ofeach; Nalge Company, A Subsidiary of Sybron, Rochester, N.Y.); Sterilewater for injection, USP (10 ml; American Pharmaceutical Partners, Inc.;Los Angeles, Calif.); Dissecting board, sterile; Sterile scalpels,forceps, and scissors, at least 2 of each; Magnetic stir bar, sterile;Receiving units, sterile (250-500 ml sizes, Nalge Company, A Subsidiaryof Sybron, Rochester, N.Y.); Funnel and metal mesh filters, sterile;Centrifuge tubes, 50 ml and 250 ml; Plastic pipets, sterile 10, 25 and50 ml; Tissue culture plates, sterile 24 and 6 well; Tissue cultureflasks, 175 cm²; Syringes, 1, 12 and 60 ml; Needles, 19 and 25 gauge;Sampling site coupler, (Baxter/Fenwal, Deerfield, Ill.); Solutiontransfer set, (Baxter/Fenwal, Deerfield, Ill.); Lifecell adapter set,(Baxter/Fenwal, Deerfield, Ill.); Interconnecting jumper tube, 8″(GIBCO, Life Technologies; Grand Island, N.Y.); Solution transfer pump;(Baxter/Fenwal, Deerfield, Ill.); Culture bags, PL732 1 liter(Baxter/Fenwal, Deerfield, Ill.); Culture bags, PL732 3 liter(Baxter/Fenwal, Deerfield, Ill.). Centrifugation g forces relate to thebottom of the centrifuge tube in a Sorvall RC-3B centrifuge: 2000rpm˜1100×g; 1500 rpm˜600×g; 800 rpm˜175×g.

Enzyme Preparation for Dispersing Tumor Cells.

Enzyme-containing medium is used to disperse tumor cells from thesurgical specimen. It is filtered through a 0.22 μm filter before useand can be stored at 4° C. for up to 3 months in a sealed container. Theenzyme-containing medium, RPMI 1640, does not have serum. It has thefollowing added components (final concentrations): antibiotics arepenicillin G (50 units/ml), Gentamicin (10 μg/ml), and Amphotericin B(1.25 μg/ml). Add the Amphotericin B after filtering the medium. Themedium contains the following final concentrations of enzymes: DNAse (30units/ml), hyaluronidase (100 μ/ml), and collagenase (1 mg/ml). Theenzyme stocks are dry powders that are stored at −20° C.

Media.

Prepare complete medium (CM) for culturing TIL by supplementingRPMI-1640 with 10% human serum (Approved source, heat-inactivated 56° C.for 30 minutes), and also with final concentrations of penicillin G (100units/ml), streptomycin (100 μg/ml), gentamicin (50 μg/ml), L-glutamine(146 μg/ml, 1 mM). Omit selected antibiotics for relevant allergies.Other GMP quality media additives may be used when necessary (e.g.imipenem). Filter CM through a 0.22 μm filter before use. Store at 4° C.and use within a week.

Dispersing Cells from Tumor Explants

On the day of tumor resection, receive the specimen in the laboratory assoon as possible after surgery. Transport the resected tissue bathed insterile saline in a sterile container. Once the specimen arrives in thelaboratory, perform all processing procedures in a laminar flowbiological safety cabinet.

Place the tissue specimen on a sterile dissecting board with alldissecting instruments (scalpels, scissors, forceps etc.) easily withinreach. The pathologist covering TIL tumor samples should be presentbefore the tissue sample is processed. Assess the extent of tumor in thespecimen. Measure the tissue dimensions using the scale in thedissection board and help the pathologist make observations needed fortheir report. Once the sampling needs of the pathologist have been metand the tumor mass has been isolated, there are several options for theinitial cell preparation for culturing tumor infiltrating lymphocytes: afine needle aspirate from the tumor tissue; tumor fragments, cut with ascalpel or scissors to ˜1-1.5 mm in each of 3 dimensions; a mechanicallydispersed, single-cell suspension; and an enzymatically generatedsingle-cell suspension.

Irrespective of the source, or sources, of the starting material,initiate the cell expansions in 24-well plates in culture mediacontaining 1000 CU/ml IL-2. When using tumor fragments, start with onefragment per well. Keep tumor fragments bathed in HBSS while they are onthe cutting board; otherwise they will dry out quickly. To prepare asingle-cell suspension mechanically, use the BD™ Medimachine System(Becton Dickinson; San Jose, Calif.).

The following description applies to enzymatically generated single cellsuspensions, but the principles are broadly applicable. Mince each tumorslice into 1-3 mm³ chunks using either cross scalpel cuts or cuttingwith scissors. Transfer up to about 30 mls of the tumor chunks with asterile plastic spoon to a sterile container with about 100 ml ofenzyme-containing medium (collagenase, hyaluronidase and DNAase). Thevolume of medium should be scaled up in a linear fashion for largeramounts of tumor. Cap the container tightly and place it on a magneticstirrer. Stir the tumor chunks gently overnight (18-24 hours) at roomtemperature. Many tumors, such as melanomas, are completely digested in2-6 hours. However, the more fibrotic tumors require overnightdigestion. In either case, the overnight digestion does not appear tohurt the final viability of the lymphocytes in the single cellsuspension. If time does not allow for an overnight digestion, tumorscan be digested for several hours at 37° C. until the cell dispersion isadequate. Also, if the overnight digestion is incomplete, an additionalfew hours of incubation at 37° C. may help.

After the tumor digestion is finished, filter the cell suspensionthrough a sterile metal mesh filter (held in a sterile plastic funnel)into 250 ml capacity sterile centrifuge tubes. The filter is designed toremove residual tumor and connective tissue. Add HBSS (Ca⁺⁺, Mg⁺⁺-free)from a fresh bottle, to the single cell suspension to top off thecentrifuge tube. Then centrifuge the single cell suspension at 1100 rpm(˜400×g) for 15 minutes. Following centrifugation, aspirate thesupernatant, resuspend the pellet in fresh HBSS, and repeat the washprocess for a total of 3 times (the first spin counting as wash number1).

Combine the cell pellets, resuspended in HBSS, in a single tube, anddetermine the total viable nucleated cell number. For records,distinguish between live tumor cells, live lymphoid cells, dead cellsand erythrocytes. Nuclei and other subcellular particles are notcounted. In general, cell viability by trypan blue exclusion is 60% orgreater, and the RBCs are less than 10× in excess of viable nucleatedcells. Otherwise, the viable nucleated cells may be enriched by using aFicoll-Hypaque gradient. Although Ficoll-Hypaque (LSM) was designed toseparate mononuclear cells from erythrocytes in peripheral blood, it isoften useful in cleaning up tumor specimens and separating dead cellsand erythrocytes from viable tumor and host lymphocytes.

All viable cells that are prepared from a patient's tumor may be set upfor TIL cultures, or, if the yield is this high, samples may beprocessed for cryopreservation. Typically, 8×10⁶ to 4×10⁸ viable cellswill be initiated as the TIL culture. Alternatively, 4×10⁶ enzymaticallydispersed cells (four wells) are cultured for TIL cultures when TIL fromtumor fragments (typically ˜8 wells) are also cultured. Enzymaticallydispersed cells may also be used to start tumor or fibroblast celllines, but it is believed that greater success is achieved whengenerating tumor lines from mechanically dispersed cells. The remainingcells may be cryopreserved in vials (2×10⁷ cells per vial are commonlyused).

In cases where Ficoll-Hypaque gradients are needed, gradients areestablished by underlaying 40 ml of the single cell suspension with 10ml of LSM. Determine the number of gradients by the packed cell volume.Use about 0.5-1.0 ml packed cells to each 50 ml FH gradient. Followingcentrifugation (2,000 rpm (˜1100×g)×15 minutes), remove the buffy coats,consolidate them in an appropriate number of 50 or 250 ml tubes, andwash 3 times using Ca⁺⁺-, Mg⁺⁺-free HBSS. For washing large tumor preps,pool the bands from 5 gradients in a single 250 ml conical. For thefirst wash, add at least an equal volume of Hanks to the cell suspensionand centrifuge at 2,000 rpm for 10 minutes. The remainingcentrifugations are at 1,500 rpm (˜600×g) for 10 minutes. After thefinal wash, assess the cell pellet for total viable cell number usingtrypan blue exclusion. For melanoma tumors, mononuclear cells can oftenbe distinguished from tumor cells by cell size and morphology.

Bulk TIL Cultures

TIL cultures are set up in 24-well sterile tissue culture plates, using5.0×10⁵ total viable nucleated cells (e.g., lymphocytes, tumor cells,macrophages, fibroblasts)/ml (1.0×10⁶ total viable cells/well, 2ml/well) of complete culture medium containing 1000 CU/ml IL-2. Theplates are incubated in a humidified incubator at 37° C., with 5% CO₂ inair.

After about one week, the TIL cultures should be assessed for growthusing trypan blue staining and counting of viable cells, and the wellsshould be viewed under a high quality inverted phase microscope. Iflymphocytes are not confluent, or the TIL have not expanded to a levelof 1.5×10⁶ TIL (lymphocytes)/ml or greater, then half of the CM isreplaced with fresh medium containing IL-2 (1000 CU/ml). This isaccomplished by removing one ml media by aspiration, taking care not todisturb the cells on the bottom of the well, and replacing it by adding1 ml fresh medium containing IL-2 (1000 CU/ml). This should be repeatedthree times per week until the culture exceeds 5×10⁶ lymphocytes/ml orbecomes nearly confluent. If the TIL are not growing by 2 weeks or theirconcentration is under 5×10⁵/ml and/or cell viability is under 60%,consider concentrating the cells and/or enriching the viable cells byFicoll-Hypaque separation.

When the TIL have grown to 1.5×10⁶/ml or greater, or if lymphocytes areconfluent throughout the wells, the individual culture wells should bepooled. Aliquots may be cryopreserved immediately or the cells may beexpanded further by re-plating at 0.7−1.5×10⁶ lymphocytes/ml in CMcontaining IL-2 (1000 CU/ml) until sufficient cells are obtained. Analiquot of bulk TIL will be examined by FACS to determine the percentageof CD8+ cells. The results of this analysis will be used to determinethe starting TIL cell number and the volume of anti-CD8 beads to be usedin the subsequent CD8 selection protocol steps.

Sufficient bulk TIL will be pooled for an estimated CD8+ cell yieldafter selection of >30×10⁶ cells. Bulk TIL cells will be subjected tothe CD8+ positive selection process as detailed in Example 9.

In process validation testing will include viable cell counting and FACSanalysis of the following samples 1) the starting bulk TIL population 2)the “flow through” depleted population and 3) the CD8+ selectedpopulation. The resulting CD8+ selected fraction will be used as theresponding cell population in clinical scale REP (see below) and willconstitute the product administered to the patient.

Clinical Scale Rapid Expansion

Each lymphocyte culture is expanded using a single Rapid ExpansionProtocol (REP). During the expansion procedure, tests for efficacy andsafety are performed as set forth in Table 3, and the cells are preparedfor patient infusion after about 14 days from REP initiation.

TABLE 3 Test Method Limits Cell viability¹ trypan blue exclusion >70%Total viable visual microscopic count >5 × 10⁸ cell number¹ TIL potency²OKT3-stimulated IFN >200 pg/ml per 10⁵ cells release Microbiologicalgram stain^(1,3) no micro-organisms seen studies aerobic culture^(3,4)no growth fungal culture^(3,4) no growth anaerobic culture^(3,4) nogrowth mycoplasma test² negative Endotoxin¹ limulus assay #5 E.U./kgPresence of Cytopathology No tumor cells per tumor cells² 200 cellsexamined ¹Performed on the final product prior to infusion. Results areavailable at the time of infusion. ²Performed 2-10 days prior toinfusion (test performed prior to final manipulation). Results areavailable at the time of infusion. ³Performed 2-4 days prior toinfusion. Results are available at the time of infusion but may not bedefinitive. ⁴Sample for test collected on the final product prior toinfusion. Results will not be available before cells are infused intothe patient.

TIL are washed by centrifugation at 600×g, resuspended in CM, counted,and viable cells are added to the other components in proportionsindicated in Table 4. Fungizone and Ciprofloxacin are added to allgrowth media starting on day 7.

TABLE 4 Component 175 cm² flask 6 × 175 cm² flask Aastrom Replicelviable TIL 1 × 10⁶   6 × 10⁶ 5-10 × 10⁶ feeder PBMC* 2 × 10⁸ 1.2 × 10⁹ 1.0 × 10⁹ OKT3  30 ng/ml  30 ng/ml  30 ng/ml rhIL-2 1000 CU/ml 1000CU/ml 1000 CU/ml CM  75 ml  450 ml  100 ml AIM V  75 ml  450 ml  100 ml*On the day that the REP is set up, previously pedigreed andcryopreserved feeder PBMC are thawed, or fresh feeder cells are preparedby Ficoll-Hypaque gradient from a lymphocytapheresis. The cells arewashed by centrifugation and resuspended in CM. Feeder cells can consistof autologous PBMC or allogeneic PBMC that have been certified to passall criteria in Table 3. Feeder cells are irradiated (40 Gy) prior touse, and radiation is carefully annotated prior to adding feeders toother REP components. PBMC, IL-2, and OKT3 are added to CM and AIM V,mixed well. Viable cells are then added and aliquots are transferred totissue culture flasks. Flasks are incubated upright at 37° C. in 5% CO₂.

On the fifth day after initiating the REP (day 5), 120 ml is aspiratedfrom each a 175 cm² flask (cells are retained on the bottom of theflask). Media is replaced with CM/AIM V 50/50 containing 1000 CU/mlIL-2.

On days 7 or 8 the cells are transferred to bags (two flasks per 3 literPL732 bag) and fed by the addition of an equal volume (300 ml) AIM Vmedia supplemented with 5% human serum, 1000 CU/ml IL-2, Fungizone (1.25mcg/ml) and Cipro (1 ml/l). The AIM-V being added to the 3 liter PL732bag is transferred with a Baxter solution transfer pump from a 10 literSTAK PACK of AIM-V medium (GIBCO; Life Technologies, Grand Island, N.Y.)using a sterile solution transfer set, a Life-adapter set, and an 8″(20.32 cm) interconnecting jumper tube.

On days 8 through harvest, the expanding cultures are maintained at acell density of 0.8−2.0×10⁶ cells per ml by the addition of Aim V mediacontaining 1000 CU/ml IL-2, Fungizone and Cipro. TIL cultures arecommonly split to new bags containing fresh medium on day 10-12. Ingeneral, REPs of bulk TIL cultures results in 500-2000 fold expansions.At any time during cell expansion, cell aliquots may be removed andassayed for quality assurance and/or sterility tests as required by thecertificate of analysis for infused cell products. If cells have grownto sufficient numbers for patient treatment, a sample is collected fromrepresentative bags or flasks for microbiology tests 2-3 days before thecells are harvested for infusion.

Preparation of Cells for Infusion

Check the quality control tests in Table 3 that are needed beforeinfusion of the cells. The product for infusion is prepared byharvesting and washing the cells in centrifuge tubes or in a continuouscentrifuge cell harvester system. Cell cultures in flasks or a smallnumber of Nexell culture bags are transferred to 250 ml centrifugetubes. These cells are centrifuged (400×g for 15 min), and thenresuspended in HBSS and combined in a single 250 ml tube. With about 4liters or more of culture fluid in Nexell culture bags, the cells areharvested with the Baxter/Fenwall harvester system, the last step ofwhich is a 2-liter wash with 0.9% sodium chloride. Cells from thecontinuous centrifuge harvest are transferred from the harvest bag to250 ml centrifuge tubes. For the last step of both harvestingprocedures, cells are centrifuged and resuspended in 100-400 ml of 0.9%sodium chloride containing 1) human albumin (25%) added to a finalconcentration of 2.5% and 2) recombinant human IL-2 at a finalconcentration of 50 CU/ml. The cell suspension is then transferred intothe infusion bag. The range of cells in the infusion bag is specified inthe clinical protocol. Aliquots are taken from the infusion bag forviable cell counting, quality control testing, and cryopreservation ofcells. The product is then transferred to the clinical team for infusionas soon as possible.

This example demonstrated the generation of young lymphocyte culturesfor adoptive transfer therapy by growth of TIL from melanoma tumors.

Example 4

This example demonstrates that tumor reactive cells are detected atequal frequencies in young TIL cultures and standard TIL culturesgenerated from TIL fragments.

The kinetics of the development of tumor reactivity in TIL cultures wasinvestigated by testing individual cultures derived from a single tumorfor activity and specificity at two times during culture progression.TIL were initially tested at the earliest time after all tumor cellswere lysed, when lymphocytes had recently become confluent in theirinitial wells. TIL cultures were tested a second time approximately 13days later after each culture had been passaged and expanded extensivelyin vitro. Eighteen of 34 consecutive tumor specimens received in theCell Processing Facility were sufficiently large to establish both youngTIL and standard TIL cultures from dissected tumor fragments (Table 5).

TABLE 5 Young Standard Specimen ID* Setup^(†) Reactive (%)^(‡) Age^(§)Setup Reactive (%) Age 4 12 10 (83) 12 3  1 (33) 27 5 24 21 (88) 17 3  3(100) 26 6 28 27 (96) 14 3  3 (100) 25 8 24 0 (0) 10 8  5 (63) 27 9 32 32 (100) 7 8  8 (100) 14 10 24 1 (4) 18 8 0 (0) NG^(¶) 13 16  2 (13) 108 0 (0) 21 16 24 0 (0) 17 8 0 (0) 33 18 16 0 (0) 14 8 0 (0) 28 22 12 0(0) 7 8  1 (13) 21 23 12  9 (75) 7 8 0 (0) 27 24 8  2 (25) 14 2  2 (100)20 25 12 0 (0) 12 8 0 (0) 27 34 12  12 (100) 12 16 12 (75) 26 Mean age12 25 Total positive (%)\\  7 (39) 7 (39) *Eighteen out of 34consecutive specimens had tumor fragments that were used to generateyoung and standard TIL. Four specimens did not expand and are notlisted. ^(†)Number of fragments set up for TIL from each tumor specimen.^(‡)Number (and percent) of TIL which expanded and had specificreactivity. Specimens which had 25% or more TIL cultures with specificreactivity as defined by at least 200 pg/mL and greater than twice IFN-γreleased by coculture with HLA-mismatched controls were consideredpositive (bold). ^(§)Age (days) at which TIL were tested for reactivity.^(¶)TIL did not expand adequately for screening. \\Total number andfrequency of positive specimens for all 18 specimens.

Specificity testing by IFN-γ release assay demonstrated that seven youngTIL from fragments had specific reactivity (39%) and seven standard TILfrom fragments also had specific reactivity (39%). Six tumor specimens(33%) had specific reactivity for both young and standard TIL. At thetime of testing for specificity, young TIL had a mean age of 12 days andstandard TIL had a mean age of 25 days.

Sixteen out of 34 consecutive tumors had enough tissue to generate bothstandard and young TIL by the enzymatic digestion method (Table 6).

TABLE 6 Young Standard Specimen ID* Setup^(†) Reactive(%)^(‡) Age^(§)Setup Reactive(%) Age 1 96 18(19) 14 4 0(0)  NG¶ 5 96 72(75) 17 3 3(100) 26 6 24 19(79) 16 3  3(100) 24 8 24 0(0) 9 4 0(0) 46 9 48 41(85)9 4 0(0) NG 10 48 1(2) 11 4 0(0) 25 16 48 0(0) 16 4 0(0) NG 18 48 0(0)13 4  2(50) 27 22 24 13(54) 13 4 0(0) 20 23 24 15(63) 13 4  2(50) 26 2448  48(100) 13 4  2(50) 19 32 24  24(100) 12 4  4(100) 24 Mean age 13 26Total positive (%)\\  7(44)  6(38) *Sixteen out of consecutive 34specimens had enzymatic digests that were used to generate young andstandard TIL. Four specimens did not expand and are not listed.^(†)Number of single-cell suspensions set up for TIL from each tumorspecimen. ^(‡)Number and percent of TIL which expanded and had specificreactivity. Specimens which had 25% or more TIL cultures with specificreactivity as defined by at least 200 pg/mL and greater than twice IFN-γreleased by coculture with HLA-mismatched controls were consideredpositive (bold). ^(§)Age (days) at which TIL were tested for reactivity.¶TIL did not expand adequately for screening. \\Total number andfrequency of positive specimens for all 16 specimens.

The same trend of reactivity observed for tumor fragments was seen forenzymatic digests of tumor. Testing by IFN-γ release assay demonstratedthat seven young TIL (44%) had specific reactivity and six standard TIL(38%) had specific reactivity. Five specimens (31%) had specificreactivity for both young and standard TIL.

This example demonstrated that tumor reactive cells are detected atequal frequencies in young TIL cultures and standard TIL culturesgenerated from TIL fragments.

Example 5

This example demonstrates that the tumor reactivity of young TIL ismaintained during in vitro expansion.

To test the stability of the tumor recognition during large numericalexpansions, four young TIL with autologous tumor reactivity (Table 7)were identified.

The TIL were expanded to approximately 2×10⁶ cells (as demonstrated byconfluent growth in 2-ml culture wells) at which time the mean age ofthese TIL was 12 days. The cells were harvested and tested forreactivity by IFN ELISA. Portions of each TIL culture were furtherexpanded with IL-2 alone (comparable to the currently used standardmethod) or rapidly expanded with IL-2, OKT3, and feeder cells for 14more days. Thus the 12-day old TIL represent young TIL prior to REP, theTIL which underwent additional standard expansion represent standard TILprior to REP (mean age=26 days), and the TIL which underwent rapidexpansion represent young TIL after REP (mean age=26 days). All TIL werethen tested for specific reactivity to autologous tumor in a singleELISA assay. The cultures that were expanded as standard TIL exhibitedan average of 19 fold growth over the 14 day assay period. Three of thefour cultures retained specific recognition of autologous tumor afterstandard expansion. The rapidly expanded TIL increased in number by anaverage of 3116 fold over the same 14-day period. The three TIL cultureswhich showed specificity with standard expansion also demonstratedspecificity after rapid expansion (Table 7).

TABLE 7 Young TIL* Standard Expansion† Rapid Expansion‡ Specimen Autol§Contr¶ Autol Contr Exp Autol Contr Exp 1 381 31 1448 111 30 1634 6504166 2 565 107 983 160 31 1053 523 3744 3 103 0 201 33 12 1599 513 28224 691 116 96 52 3 424 1021 1733 *TIL generated from four enzymaticallydigested tumor samples were tested for specific reactivity when eachculture expanded to 2 × 10⁶ lymphocytes (mean = 12 days). †Young TILwere expanded using standard methods for 14 more days and then tested.‡Young TIL were rapidly expanded for 14 more days and tested. §Valuesindicate IFN-γ release (pg/mL) when cocultured with autologous tumor.Bold numbers indicate a tumor-specific reactive test defined as at least100 pg/mL and greater than twice IFN-γ released by coculture withHLA-mismatched controls. ¶Values indicate IFN-γ release (pg/mL) whencocultured with HLA-mismatched control tumors.

As shown in Table 7, TIL demonstrating specific reactivity at a youngage usually maintain reactivity after both standard expansion and rapidexpansion, and supports the use of young TIL instead of standard TIL foradoptive transfer.

This example demonstrated that the tumor reactivity of young TIL ismaintained during in vitro expansion.

Example 6

This example demonstrates that young TIL have a higher frequency of CD4+cells than standard TIL.

To investigate the cellular composition of TIL cultures over time,aliquots of 14 TIL cultures were sampled and cryopreserved as soon asthe lymphocytes became confluent and tumor cells were eliminated fromwells. Then cultures were maintained by standard methods for 14additional days and another sample was cryopreserved. Then both sampleswere thawed and analyzed by FACS simultaneously. The mean age was 15days for young TIL and 31 days for standard TIL. Analysis of lymphocytesubsets in the TIL demonstrated that the young TIL had a higherfrequency of CD4⁺ lymphocytes than standard TIL (FIG. 1A, p=0.02).However, there were no significant differences in the frequency of CD8⁺cells between young and standard TIL (FIG. 1B). The frequency ofCD3-CD56⁺ natural killer (NK) cells was lower in young TIL (p=0.02, FIG.1C).

This example demonstrated that young TIL have a higher frequency of CD4⁺cells than standard TIL.

Example 7

This example demonstrates that young TIL express higher levels ofcostimulatory molecules than standard TIL.

FACS analysis of gated CD8⁺ cells from the TIL populations generated inExample 6 demonstrated a strong relationship between culture age and theexpression of CD27 and CD28 (FIGS. 2A, 2B, and 2C). Strikingly, thepercentage of CD8⁺ cells co-expressing CD27 and CD28 was higher in all14 samples of younger TIL than standard TIL. There were no significantdifferences in the expression of CD62L and CCR7 by young and standardTIL (data not shown).

This example demonstrated that young TIL express higher levels ofcostimulatory molecules than standard TIL.

Example 8

This example demonstrates that young TIL have longer telomeres thanstandard TIL.

To investigate the impact of culture time on the telomere lengths of theTIL, 495 independent TIL cultures from 48 consecutive specimens weretested. Telomere lengths of 495 TIL from 48 consecutive patients wereevaluated by quantitative fluorescent in-situ hybridization. As shown inFIG. 3, although the telomere lengths varied widely at any given TILage, there was an inverse correlation between time in culture and themean telomere length of TIL (p<0.001).

This example demonstrated that young TIL have longer telomeres thanstandard TIL.

Example 9

This example demonstrates the enrichment of cultured T cells for CD8⁺cells.

The materials used in this Example include: Peripheral Blood Lymphocyte(PBL) product containing up to 40×10⁹ total cells and up to 4×10⁹ CD8⁺cells; CliniMACS CD8 MicroBeads; CliniMACS^(plus) Instrument, MiltenyiBiotec (e.g., Order No. 155-02), software version 2.3×; 1 CliniMACSTubing Set, Miltenyi Biotec (e.g., Order No. 165-01), or 1 CliniMACS LSTubing Set, Miltenyi Biotec (e.g., Order No. 168-01); 1 Pre-SystemFilter, Miltenyi Biotec, Order No. 181-01; 1 Luer/Spike Interconnector,Miltenyi Biotec, Order No. 187-01; CliniMACS PBS/EDTA buffer, MiltenyiBiotec (e.g., Order No. 705-25); Human Serum Albumine (HSA) supplementto CliniMACS PBS/EDTA buffer, final concentration 0.5%; 250 mlcentrifuge tubes; Immune Globulin Intravenous (Human), 10% (GAMMAGARDLIQUID, Baxter); Transfer Bags 600 ml, Miltenyi Biotec, Order No.190-01; Digital Balance; Sterile Tubing Welder; Orbital Shaker; SamplingSite Coupler; Tubing Slide Clamps or Scissor clamps. CliniMACS CD8MicroBeads are manufactured under an ISO 9001 certified Quality Systemand follows cGMP guidelines.

A typical pheresis product was obtained and processed (starting cells).3.88×10⁹ peripheral blood mononuclear cells were applied to theCliniMACS^(plus) and Enrichment 1.1 program using tubing set 165-01,Process CODE: 0211020F0000834.

The positive selection of CD8 positive T cells is performed byimmunomagnetic labeling of CD8 expressing cells and enrichment of thesecells from the target fraction by automatic cell separation using theCliniMACS^(plus) Instrument. The highly purified CD8+ cells arecollected in the Cell Collection Bag.

The content of one vial of CliniMACS CD8 MicroBeads is sufficient forlabeling of up to 4×10⁹ CD8 positive cells out of a total leukocytenumber of up to 40×10⁹ cells (normal scale preparation (A). Forprocessing of 2 to 4×10⁹ CD8 positive cells or 20 to 40×10⁹ leukocytestwo CliniMACS CD8 MicroBeads vials are needed to achieve sufficientlabelling (large scale preparation (B)). Weigh the empty CellPreparation tube prior to transferring the PBL into the Cell Preparationtube. Determine the volume of the PBL by weighing the filled CellPreparation tube and subtracting the empty tube weight. Use a smallaliquot of the PBL to determine the total number of leukocytes and theviability. When process timing allows, use FACS analysis to determinethe percentage of target cells. If process timing is insufficient forFACS analysis, assume a CD8+ percentage of 20% of the total pheresisproduct.

Dilute the PBL (1:3) with CliniMACS PBS/EDTA Buffer (supplemented with0.5% HSA) and centrifuge the cells at 300×g for 10 minutes. Calculatethe amount of buffer to be added using the following equation: Weight ofbuffer to be added (g)=Weight of PBL (g)×2. Spin down the cells (300×g,10 min, room temperature (+19° C. to +25° C.)). Remove the supernatantand adjust the sample to a labeling volume of A) 400 mL (normal scale)and distribute evenly to two 250 ml tubes. Add 1.25 ml Gammagard pertube (2.5 ml total). Mix gently. Add 1.0 ml CliniMACS CD8 MicroBeads toeach tube and mix carefully.

Incubate the cell preparation tubes for 30 minutes at controlled roomtemperature (+19° C. to +25° C.) on an orbital shaker at 25 rpm. After30 minutes rocking, spin down the cells for 15 minutes at roomtemperature and 300×g. Remove as much supernatant as possible from theCell Preparation tubes and resuspend the cells in CliniMACS buffer.Combine the cell pellets into one tube. Adjust the cell concentrationafter the washing step to 0.4×10⁹ total cells/mL. The final samplingvolume of the PBL for loading on the CliniMACS^(plus) Instrument shouldnot exceed 275 mL.

Transfer a 0.5 mL sample to a sample tube for flow cytometric analysis.It is recommended to determine at least the cell concentration, theviability, and the frequency/number of the target cells.

Automated Separation

Switch on the CliniMACS^(plus) Instrument and select a suitable program:positive selection of cells, ENRICHMENT 1.1 is recommended.

Confirm your choice by pressing “ENT” and select a tubing set. CliniMACSTubing Sets are available with different tubing and column geometry.They differ, e.g., in the maximum cell capacity that can be processed.For further information refer to the CliniMACS^(plus) User Manual“General Instructions.” Enter the Order No. of the selected tubing set.Selection program ENRICHMENT 1.1, is “staged loading” programs. Theyinclude a query for the following parameters to adjust the selectionsequence to each individual sample and to provide information on therequired buffer and bag volumes: WBC concentration; percentage oflabeled cells; total volume of the sample ready for loading on theCliniMACS Tubing Set.

Follow the instructions given on the instrument screen and connect anappropriate bag to the tubing set using a Luer/Spike Interconnector(Order No. 187-01). Ensure that the slide clamp of the Luer/SpikeInterconnector is open. If more than 1 L of buffer is needed, connecttwo buffer bags using a Plasma Transfer Set with two couplers (order No.186-01). Use the second port of one of the buffer bags for theconnection to the tubing set.

Follow the instructions on the instrument screen for the installation ofthe tubing set and start the automated separation program. To ensureproduct sterility, all cell preparation and manipulation steps areperformed in a laminar flow hood under aseptic conditions, in the samelaboratory where the cells will later be transduced and cultured foreventual production of the patient-specific cell therapeutics.

After the separation has been finished, determine the weight of CellCollection Bag and take a sample for flow cytometry analysis. Anadditional analysis of non-target fractions (negative and buffer wastefraction) is useful for a further process optimization.

0.69×10⁹ CD8 enriched cells were recovered and analyzed by FACS analysis(Table 8).

TABLE 8 CD3⁺ Starting CD8 enriched lymphocytes cells (%) cells (%) CD3⁺CD4⁺ 33.35 5.02 CD3⁺ CD8⁺ 44.10 88.67 CD3⁻ CD8⁺ 5.91 10.34

This example demonstrated the enrichment of cultured T cells for CD8⁺cells.

Example 10

This example demonstrates the transduction efficiency of CD8 enrichedcells following CliniMACS^(plus) separation.

Samples from the initial (unseparated) PBMC pheresis product and theCD8⁺ enriched product obtained in Example 9 were stimulated for two dayswith OKT3 and IL-2 under optimized conditions.

Samples were transduced with clinical grade MART TCR retroviralsupernatant and expanded for four more days. Untransduced controls (NV)were expanded similarly. All cultures expanded at equivalent rates.

Transduction efficiency was determined by FACS analysis for VB12expression and A2/MART-1 tetramer staining (Table 9).

TABLE 9 Starting cells (%) CD8 enriched cells (%) NV control MART TCR NVControl MART TCR MART-1 tetramer staining CD8+ MART− 53.1 55.5 95.6 96.4CD8+ MART+ 0.2 0.4 0.2 1.3 TCR VB12 expression CD8+ VB12− 54.0 44.6 97.568.2 CD8+ VB12+ 0.4 13.6 1.1 30.9

This example demonstrated the transduction efficiency of CD8 enrichedcells following CliniMACS^(plus) separation.

Example 11

This example demonstrates that (i) culturing autologous T cells; (ii)rapidly expanding the cultured T cells; (iii) administering to themammal nonmyeloablative lymphodepleting chemotherapy; and (iv) afteradministering nonmyeloablative lymphodepleting chemotherapy,administering to the mammal the expanded T cells, wherein the T cellsadministered to the mammal are about 19 to about 35 days old, promotesthe regression of cancer in human patients.

Objectives.

In cohort 1, to determine the ability of autologous TIL cells infusedafter minimal in vitro culture in conjunction with high dose aldesleukin(IL-2) following a non-myeloablative lymphodepleting preparative regimento mediate tumor regression in patients with metastatic melanoma.

In cohort 2, to determine the ability of autologous CD4⁺ cell depletedTIL cells infused after minimal in vitro culture in conjunction withhigh dose aldesleukin (IL-2) following a non-myeloablativelymphodepleting preparative regimen to mediate tumor regression inpatients with metastatic melanoma.

In cohort 3, to determine the ability of autologous CD4⁺ cell depletedTIL cells infused after minimal in vitro culture in conjunction withhigh dose aldesleukin following chemoradiation lymphoid depletingregimen to mediate complete tumor regression in patients with metastaticmelanoma.

Evaluate the toxicity of these treatment regimens.

Determine the rate of repopulation of the young TIL cells in treatedpatients and establish in vitro correlates of TIL cultures that mediateobjective response and in vivo persistence.

Design:

Patients will undergo resection to obtain tumor for generation ofautologous TIL cultures.

In Cohort 1, all patients will receive a non-myeloablative lymphocytedepleting preparative regimen of cyclophosphamide (60 mg/kg/day IV) ondays −7 and −6 and fludarabine (25 mg/m²/day IV) on days −5 through −1.On day 0 patients will receive the infusion of autologous TIL and thenbegin high-dose aldesleukin (720,000 IU/kg IV every 8 hours for up to 15doses). Clinical and Immunologic response will be evaluated about 4-6weeks after TIL infusion.

In Cohort 2, CD4⁺ cells will be eliminated from the cultures, using theMiltenyi CliniMACS^(plus) apparatus, prior to performing the rapidexpansion of the young TIL cells. Patients in cohort 2 will receive CD4⁺cell depleted young unselected TIL. Patients will also receive high doseIL-2 after non-myeloablative but lymphodepleting chemotherapypreparative regimen as described above for cohort 1. Clinical andimmunologic response will be evaluated about 4-6 weeks after TILinfusion. Using a small optimal two-stage Phase II design, initially 18patients will be enrolled, and if three or more of the first 18 patientshave a clinical response (PR or CR), accrual will continue to 35patients, targeting a 30% goal for objective response.

In Cohort 3, patients will receive a chemoradiation lymphocyte depletingpreparative regimen consisting of cyclophosphamide, fludarabine, and 600cGy total body irradiation followed by intravenous infusion ofautologous CD4⁺ cell depleted young TIL plus IV high dose IL-2. Clinicaland immunologic response will be evaluated about 4-6 weeks after TILinfusion.

Eligibility Assessment and Enrollment 2.1 Eligibility Criteria

2.1.1 Inclusion Criteria

Inclusion criteria are set forth in Table 10.

2.1.2 Exclusion Criteria

Exclusion criteria are set forth in Table 10.

TABLE 10 Inclusion Criteria Exclusion Criteria Measurable metastaticmelanoma with at Women of child-bearing potential who are least onelesion that is resectable for TIL pregnant or breastfeeding because ofthe generation potentially dangerous effects of the preparativechemotherapy on the fetus or infant. Patients with one or more brainmetastases Systemic steroid therapy required. less than 1 cm each, andany patients with 1 or 2 brain metastases greater than 1 cm must havebeen treated and stable for 3 months Greater than or equal to 18 yearsof age Active systemic infections, coagulation disorders or other activemajor medical illnesses of the cardiovascular, respiratory or immunesystem, as evidenced by a positive stress thallium or comparable test,myocardial infarction, cardiac arrhythmias, obstructive or restrictivepulmonary disease. Willing to practice birth control during Any form ofprimary immunodeficiency treatment and for four months after receiving(such as Severe Combined the preparative regimen ImmunodeficiencyDisease and AIDS). Life expectancy of greater than three monthsOpportunistic infections (The experimental treatment being evaluated inthis protocol depends on an intact immune system. Patients who havedecreased immune competence may be less responsive to the experimentaltreatment and more susceptible to its toxicities.) Willing to sign adurable power of attorney History of severe immediate hypersensitivityreaction to any of the agents used in this study. Able to understand andsign the Informed History of coronary revascularization or ConsentDocument ischemic symptoms Clinical performance status of Eastern Anypatient known to have an LVEF less Cooperative Oncology Group (ECOG) 0or 1. than or equal to 45%. Hematology: Documented left ventricularejection fraction Absolute neutrophil count greater than (LVEF) of lessthan or equal to 45% tested in 1000/mm³ without support of filgrastimpatients with: Normal white blood cell (WBC) Clinically significantatrial and/or ventricular (>3000/mm³). arrhythmias including but notlimited to: Hemoglobin greater than 8.0 g/dl atrial fibrillation,ventricular tachycardia, Platelet count greater than 100,000/mm³ secondor third degree heart block Age ≧60 years old Serology: DocumentedForced Expiratory Volume in Seronegative for human immunodeficiency OneSecond (FEV1) less than or equal to virus (HIV) antibody. (Theexperimental 60% predicted tested in patients with: treatment beingevaluated in this protocol A prolonged history of cigarette smokingdepends on an intact immune system. Symptoms of respiratory dysfunctionPatients who are HIV seropositive can have decreased immune competenceand thus be less responsive to the experimental treatment and moresusceptible to its toxicities.) Seronegative for hepatitis B orhepatitis C. Chemistry: Patients will be excluded from cohort 3 (butSerum alanine aminotransferase eligible for cohort 2), if any of thefollowing (ALT)/asparatate aminotransferase (AST) conditions occur: lessthan three times the upper limit of Prior radiation which makes thepatient normal. ineligible to receive 600 cGy. Serum creatinine lessthan or equal to 1.6 mg/dl. Inability to mobilize CD34⁺ cells as Totalbilirubin less than or equal to 2 mg/dl, described herein. except inpatients with Gilbert's Syndrome who must have a total bilirubin lessthan 3 mg/dl. More than four weeks must have elapsed since any priorsystemic therapy at the time the patient receives the preparativeregimen, and patients' toxicities must have recovered to a grade 1 orless (except for toxicities such as alopecia or vitiligo). Patients mayhave undergone minor surgical procedures with the past 3 weeks, as longas all toxicities have recovered to grade 1 or less or as specified inthe eligibility criteria herein. Six weeks must have elapsed since anyprior anti-CTLA4 antibody therapy to allow antibody levels to decline.Patients who have previously received any anti-CTLA4 antibody must havea normal colonoscopy with normal colonic biopsies.

2.2 Research Eligibility Evaluation

Within 4 weeks prior to starting the chemotherapy regimen, evaluationswill be performed as set forth in Table 11.

TABLE 11 Evaluation Complete physical examination including height,weight and vital signs and eye exam, noting in detail the exact size andlocation of any lesions that exist. Chest x-ray electrocardiogram (EKG)Baseline CT of the chest, abdomen and pelvis, and brain magneticresonance imaging (MRI) to evaluate the status of disease. Additionalscans and x-rays may be performed if clinically indicated based onpatients' signs and symptoms. Cardiac Multi Gated Acquisition Scan(MUGA) or echocardiogram, stress thallium) and pulmonary evaluation(PFTs) and colonoscopy. Note: cardiac evaluation may be performed up to6 months prior to treatment. HIV antibody titer and HbsAG determination,anti hepatitis C virus (HCV), anti cytomegalovirus (CMV) antibody titer,herpes simplex virus (HSV) serology, and Epstein-Barr virus (EBV) panelVerification that HLA typing is completed For patients in cohort 3 only,15 mL of clean catch urine to be tested

Within 14 days prior to starting the chemotherapy regimen, evaluationswill be performed as set forth in Table 12:

TABLE 12 Evaluation Chem 20 [Sodium (Na), Potassium (K), Chloride (Cl),Total CO₂(bicarbonate), Creatinine, Glucose, Urea nitrogen (BUN),Albumin, Calcium total, Magnesium total (Mg), Inorganic Phosphorus,Alkaline Phosphatase, Alanine Aminotransferase/Glutamic-PyruvicTransaminase (ALT/GPT), Aspartate aminotransferase/Glutamyl oxaloacetictransaminase (AST/GOT), Total Bilirubin, Direct Bilirubin, LD, TotalProtein, Total creatine kinase (CK), Uric Acid]and thyroid bloodchemistry panel. complete blood count (CBC), differential, Prothrombintime/partial thromboplastin time (PT/PTT), platelet count Urinalysis andculture, if indicated

Within 7 days prior to starting the chemotherapy regimen, a β-HCGpregnancy test (serum or urine) on all women of child-bearing potentialwill be performed.

2.3 Patient Registration

Patients will be registered by the clinical fellow or research nurse atthe time that they enter the study by faxing a completed eligibilitychecklist to the Central Registration Office (CRO). Written confirmationof registration to the protocol will be obtained from CRO and placed onthe patient's research record.

Study Implementation Study Design: 3.1.1 Pre-Treatment Phase

3.1.1.1 Autologous Stem Cell Collection (for Cohort 3 only)

Prior to the treatment phase of this study, patients will have stemcells collected and stored for re-infusion after the myeloablation andcell therapy. Patients will receive filgrastim at 8 mcg/kg dose BID(total=16 mcg/kg/day) by subcutaneous injection. The morning dose offilgrastim will be given at approximately 7:00 am each day. Stem cellswill be collected by apheresis. Autologous apheresis will start on thefifth day of filgrastim administration, and filgrastim administrationand apheresis will be repeated daily for a maximum of 2 days to achievea sufficient dose of CD34⁺ cells after ex vivo processing (MiltenyiClinicMACs). For each daily apheresis a blood volume of 20-35 liters ofblood will be processed, using ACD-A anticoagulation, peripheral orcentral venous access, and calcium replacement as needed, per standardoperating procedure of the NIH Clinical Center DTM. Sufficient CD34⁺cell doses after Miltenyi ClinicMACs positive selection are defined as aTARGET of >4×10⁶/kg recipient weight, and a minimum of >2×10⁶/kgrecipient weight. Patients who do not reach the minimum dose of selectedCD34⁺ cells after 2 apheresis collections may be considered for a secondcycle of filgrastim mobilization and apheresis collections or may beconsidered for bone marrow harvest.

If the minimum dose of CD34⁺ cells has not been reached after 2 cyclesof mobilization and collection and/or bone marrow harvest, the patientwill be treated in cohort 2.

CD34⁺ cells will be processed and cryopreserved according to DTM policyand procedure until needed for cell infusion.

3.1.1.2 Cell Preparation (for All Cohorts)

Treatment will be similar to that in approved protocol99-C-0158/T99-0078: Treatment of Patients with Metastatic Melanoma usingCloned Lymphocytes Following Administration of a Nonmyeloablative butLymphocyte Depleting Regimen. Patients with metastatic melanoma willhave TIL obtained while enrolled on the Surgery Branch protocol03-C-0277, “Cell Harvest and Preparation for Surgery Branch AdoptiveCell Therapy Protocols”. Separate tumor biopsies may be performed toobtain TIL for subsequent lymphocyte cultures. TIL will be grown andexpanded for this trial generally as described in Example 3, and the age(in days) of the cells administered to each of 25 patients is asfollows: 20, 24, 26, 26, 26, 26, 27, 27, 28, 28, 29, 29, 29, 29, 32, 34,34, 34, 34, 34, 35, 35, 35, 36, and 36. The average age of the cells is30 days. As in the prior cell infusion protocols, volunteers on theSurgery Branch protocol 03-C-0277, “Cell Harvest and Preparation forSurgery Branch Adoptive Cell Therapy Protocols” will donate whole bloodand serum to be isolated and used in cell culture according to thestrict safety criteria outlined in 03-C-0277. Volunteers will alsoundergo apheresis to obtain mononuclear cells to be used as feeder cellsin cell culture. The procedures used are those in routine use in theDepartment of Transfusion Medicine in the Clinical Center. Separateconsents will be obtained from all blood and apheresis volunteers.

Prior to patient enrollment on this study, TIL cultures will bemonitored regularly, and at the earliest feasible time, TIL will beassessed for potency by OKT3-stimulated IFN release (greater than 200pg/ml per 10⁵ cells). Once cells have been deemed eligible for use inthis trial, patients will be consented on this study and enrolled. Thepatient must meet the eligibility criteria prior to administration ofthe preparative regimen. Growth and expansion of the final product willbe performed after the patient has consented to participate in thisspecific study.

3.1.2 Treatment Phase

Once cells exceed the potency requirement and are projected to exceed5×10⁸ cells (measured by visual microscopic count), (approximately 7days after the REP procedure has been initiated) the patient willreceive the lymphocyte depleting preparative regimen consisting offludarabine and cyclophosphamide (in cohorts 1 and 2), followed byinfusion of up to 3×10″ lymphocytes (minimum of 1×10⁹) and theadministration of aldesleukin. In cohort 3, patients will receive alymphocyte depleting preparative regimen consisting of cyclophosphamideand fludarabine plus 600 cGy total body irradiation followed in one tofour days by intravenous infusion of up to 3×10″ lymphocytes (minimum of1×10⁹) and the administration of aldesleukin.

Approximately 4-6 weeks after cell infusion, patients will undergo acomplete tumor evaluation and evaluation of toxicity and immunologicparameters. This will comprise one course of therapy. Patients willreceive no other experimental agents while on this protocol.

Drug Administration:

3.2.1 For Cohorts 1 (closed) and 2: Preparative Regimen withCyclophosphamide and Fludarabine

On Day −7 and −6, patients will be treated as set forth in Table 13.

TABLE 13 Time Treatment  1 am Hydrate: Begin hydration with 0.9% SodiumChloride Injection containing 10 meq/L of potassium chloride at 2.6ml/kg/hr (starting 11 hours pre-cyclophosphamide and continue hydrationuntil 24 hours after last cyclophosphamide infusion) 11 am Ondansetron(0.15 mg/kg/dose [rounded to the nearest even mg dose between 8 mg and16 mg based on patient weight] IV every 8 hours × 3 days) will be givenfor nausea. Furosemide 10-20 mg iv. 12 pm (noon) Cyclophosphamide 60mg/kg/day × 2 days IV in 250 ml D5W with mesna 15 mg/kg/day × 2 daysover 1 hr. If patient is obese (BMI > 35) drug dosage will be calculatedusing practical weight.  1 pm Begin to monitor potassium level every 12hours until hydration is stopped. KCl will be adjusted to maintain serumpotassium levels in the normal range.  1 pm Begin mesna infusion at 3mg/kg/hour intravenously diluted in a suitable diluent over 23 hoursafter each cyclophosphamide dose. If patient is obese (BMI > 35) drugdosage will be calculated using practical weight.

On Day −5, IV hydration is stopped (24 hours after last cyclophosphamidedose). If urine output is less than 1.5 ml/kg/hr, an additional 20 mgfurosemide iv is given. If body weight is greater than 2 kg over precyclophosphamide value, additional furosemide 20 mg iv is given.

On Day −5 to Day −1, Fludarabine 25 mg/m²/day IVPB is given daily over30 minutes for 5 days. If patient is obese (BMI>35) drug dosage will becalculated using practical weight.

3.2.2 Cell Administration (for Cohorts 1 (closed) and 2):Day 0 (One to Four Days after the Last Dose of Fludarabine):

Cells will be infused intravenously (i.v.) on the Patient Care Unit over20 to 30 minutes (between one and four days after the last dose offludarabine). Cell infusions will be given as an inpatient.

Aldesleukin 720,000 IU/kg IV (based on total body weight) over 15 minuteevery eight hours beginning within 24 hours of cell infusion andcontinuing for up to 5 days (maximum of 15 doses.)

Day 1-4 (Day 0 is the Day of Cell Infusion):

Beginning on day 1 or 2, filgrastim will be administered subcutaneouslyat a dose of 5 mcg/kg/day (not to exceed 300 mcg/day). Filgrastimadministration will continue daily until neutrophil count >1.0×10⁹/L×3days or >5.0×10⁹/L.

Aldesleukin 720,000 IU/kg IV (based on total body weight) over 15minutes is given every eight hours for up to 5 days.

3.2.3 For Cohort 3 only: Preparative Regimen with Cyclophosphamide,Fludarabine and TBI

On Day −6 and −5, patients will be treated as set forth in Table 13.

On Day −6 to Day −2, Fludarabine 25 mg/m²/day IVPB is administered dailyover 15-30 minutes for 5 days. If the patient is obese (BMI >35) drugdosage will be calculated using practical weight. (The fludarabine willbe started approximately 1-2 hours after the cyclophosphamide and mesnaon Days −6 and −5). To allow as much time between the last dose offludarabine and the administration of the cell treatment, the timing ofthe fludarabine administration can be moved to an hour earlier eachday.)

On Day −4, IV hydration is stopped (24 hours after last cyclophosphamidedose). If urine output <1.5 ml/kg/hr, additional 20 mg furosemide iv isgiven. If body weight >2 kg over pre cyclophosphamide value giveadditional furosemide 20 mg iv.

On Day −2 and Day −1, prior to TBI, patients will receive a single doseof IV ondansetron (ondansetron 0.15 mg/kg IV [rounded to the nearesteven mg dose between 8 mg and 16 mg based on patient weight]×1 dosepre-TBI). Patients will receive 2Gy of TBI (see Sec 3.2.5) twice on day−2 and once on day −1 (total dose 6 Gy) at a rate of 0.07 Gy/minuteusing a linear accelerator in Radiation Oncology.

3.2.4 Cell Administration Cohort 3 Only)

Day 0 (One to Three Days after the Last Dose of TBI):

CD4⁺ depleted young TIL cells will be infused intravenously (i.v.) onthe Patient Care Unit over 20 to 30 minutes. Cell infusions will begiven as an inpatient. Aldesleukin 720,000 IU/kg IV (based on total bodyweight) over 15 minute every eight hours beginning within 24 hours ofcell infusion and continuing for up to 5 days (maximum of 15 doses.).Beginning on day 0 or 1, filgrastim will be administered subcutaneouslyat a dose of 5 mcg/kg/day (not to exceed 300 mcg/day). Filgrastimadministration will continue daily until neutrophil count >1.0×10⁹/L×3days or >5.0×10⁹/L.

Day 1: (Day 0 is the day of TIL Infusion)

The cryopreserved autologous CD34⁺ selected stem cell product will bethawed and administered intravenously immediately. The minimum dose willbe >2×10⁶ CD34⁺ cells per kg. CD34⁺ cells are returned on day 1 tomaximize the exchange only of the transferred T cells (day 0) to thehomeostatic environment resulting from lymphodepletion.

Day 1-4:

Aldesleukin 720,000 IU/kg IV (based on total body weight) over 15minutes every eight hours for up to 5 days.

3.2.5 Total Body Irradiation (TBI) (for Cohort 3 Only)

All patients should be treated with a linear accelerator using energieshigher than 4MV. It is anticipated that TBI will be delivered on day −2,−1. TBI will be delivered with lateral fields using extended SSD/SADvalues of 200-500 cm (depending on machine/vault size). Tissuecompensation for the lung and head and neck regions may be employed tomaximize dose homogeneity. Patients will be treated with TBI to a totaldose of 600 cGy delivered in 200 cGy fractions twice on day −2, at least6 hours apart and once on day −1.

Occasionally, the total dose/technique of TBI may require modificationsdue to patient factors (unexpected or severe (grade 4-5) adverse events,serious medical illnesses not conducive to stable patient transfer,patient refusal, etc.) or treatment factors (linear accelerator machineoffline, etc.). Modifications to the radiation treatment will be at thediscretion of the treating radiation oncologist and will be discussedwith the Principal Investigator or Study Chairperson.

3.2.6 Infection Prophylaxis (all Cohorts) 3.2.6.1 Pneumocystis CariniiPneumonia

All patients will receive the fixed combination of trimethoprim andsulfamethoxazole [SMX] as double strength (DS) tab (DS tabs=TMP 160mg/tab, and SMX 800 mg/tab) P.O. daily three times a week onnon-consecutive days, beginning on day −6 and stopping when the CD4count is above 200 and for at least 6 months post chemotherapy.

Pentamidine will be substituted for TMP/SMX-DS in patients with sulfaallergies. It will be administered aerosolized at 300 mg per nebulizerwithin one week prior to admission and continued monthly until CD4 countis above 200 and for at least 6 months post chemotherapy.

3.2.6.2 Herpes Virus Prophylaxis

Patients with positive HSV serology will be given valacyclovir orally ata dose of 500 mg daily the day after chemotherapy ends, or acyclovir,250 mg/m² IV q 12 hrs if the patient is not able to take medication bymouth which is continued until absolute neutrophil count is greater than1000/mm³. Reversible renal insufficiency has been reported with IV butnot oral acyclovir. Neurologic toxicity including delirium, tremors,coma, acute psychiatric disturbances, and abnormal EEGs have beenreported with higher doses of acyclovir. Should this occur, a dosageadjustment will be made or the drug will be discontinued. Acyclovir willnot be used concomitantly with other nucleoside analogs which interferewith DNA synthesis, e.g., ganciclovir. In renal disease, the dose isadjusted as per product labeling.

3.2.6.3 Fungal Prophylaxis (Fluconazole)

Patients will start Fluconazole 400 mg p.o. the day after chemotherapyconcludes and continue until the absolute neutrophil count is greaterthan 1000/mm³. The drug may be given IV at a dose of 400 mg in 0.9%sodium chloride USP daily in patients unable to take it orally.

3.2.6.4 Empiric Antibiotics

Patients will start on broad-spectrum antibiotics, either a 3^(rd) or4^(th) generation cephalosporin or a quinolone for fever of 38.3° C.once or two temperatures of 38.0° C. or above at least one hour apart,and an ANC <500/mm³. Aminoglycosides should be avoided unless clearevidence of sepsis. Infectious disease consultation will be obtained forall patients with unexplained fever or any infectious complications.

3.2.7 Blood Product Support (for all Cohorts)

Using daily CBCs as a guide, the patient will receive platelets andpacked red blood cells (PRBC's) as needed. Attempts will be made to keepHb >8.0 gm/dl, and pits >20,000/mm³. Note, patients with brainmetastasis will be transfused when platelets fall below 50,000/mm³. Allblood products with the exception of the stem cell product will beirradiated. Leukocyte filters will be utilized for all blood andplatelet transfusions to decrease sensitization to transfused WBCs anddecrease the risk of CMV infection.

3.2.8 Aldesleukin: Intravenous Administration (for all Cohorts)

Aldesleukin will be administered at a dose of 720,000 IU/kg (based ontotal body weight) as an intravenous bolus over a 15 minute period everyeight hours beginning on the day of cell infusion and continuing for upto 5 days (maximum 15 doses). Doses may be skipped depending on patienttolerance. Doses will be skipped if patients reach Grade III or IVtoxicity due to aldesleukin except for the reversible Grade IIItoxicities common to aldesleukin such as, for example, diarrhea, nausea,vomiting, hypotension, skin changes, anorexia, mucositis, dysphagia.Toxicities will be managed. If these toxicities can be easily reversedwithin 24 hours by supportive measures then additional doses may begiven. Additional instances may arise when in the clinical judgment ofthe attending physician, based on the extensive clinical experience inthe Surgery Branch with aldesleukin, when doses of aldesleukin may beskipped. If greater than 2 doses of aldesleukin are skipped, aldesleukinadministration will be stopped. Aldesleukin will be administered as aninpatient and will be purchased by the NIH Clinical Pharmacy Departmentfrom commercial sources.

3.3 On-Study Evaluation

Within 14 days prior to starting the preparative regimen, patients willhave a complete blood count, electrolytes, BUN, creatinine, liverfunction tests and serum chemistries performed. If any results arebeyond the criteria established for eligibility, the patient will notproceed until the abnormalities can be resolved.

For patients in cohort 3 only, 15 mL of clean catch urine will becollected to be tested.

During the Preparative Regimen: DAILY

Patients will be evaluated on the basis of: Complete Blood Count; Chem20: [Sodium (Na), Potassium (K), Chloride (Cl), Total CO₂ (bicarbonate),Creatinine, Glucose, Urea nitrogen (BUN), Albumin, Calcium total,Magnesium total (Mg), Inorganic Phosphorus, Alkaline Phosphatase,ALT/GPT, AST/GOT, Total Bilirubin, Direct Bilirubin, LD, Total Protein,Total CK, Uric Acid]; and Urinalysis. CMV antigen assay will be assessedif clinically indicated (e.g., unexplained fevers, pulmonary changes).For patients in cohort 3 only, 15 mL of clean catch urine to be testedwill be collected at one week after TBI, approximately 1 month after TBIand 6 months after TBI.

After Cell Infusion

After cell infusion vital signs will be monitored hourly until stableand then routinely (every 4 hours) unless otherwise clinicallyindicated.

During and after Aldesleukin Administration (Until HospitalDischarge)—Every 1-2 Days

Patients will be evaluated by Complete Blood Count and Chem 20: [Sodium(Na), Potassium (K), Chloride (Cl), Total CO2 (bicarbonate), Creatinine,Glucose, Urea nitrogen (BUN), Albumin, Calcium total, Magnesium total(Mg), Inorganic Phosphorus, Alkaline Phosphatase, ALT/GPT, AST/GOT,Total Bilirubin, Direct Bilirubin, LD, Total Protein, Total CK, UricAcid].

3.3.5 Additional Research Evaluations

Once total lymphocyte count is greater than 200/mm³, the followingsamples will be drawn and sent to the TIL lab on Monday, Wednesday andFriday: 5 CPT tubes (10 ml each) and 1 SST tube (10 ml).

Biopsies of tumor tissue or lymph node may be performed but are notrequired during the course of therapy. Studies will be performed toevaluate the antigen expression by the tumor and to evaluate thereactivity of lymphocytes grown from these biopsies. Biopsies will beperformed at baseline, after the course of therapy, and in the event ofresponse. These biopsies will only be performed if minimal morbidity isexpected based on the procedure performed and the granulocyte andplatelet count.

Peripheral blood lymphocytes (PBL) will be purified by centrifugation ona Ficoll cushion, then evaluated for function and phenotype. Lymphocytesmay be tested by cytolysis assays, cytokine release, limiting dilutionanalysis and by other experimental studies. Immunological monitoringwill consist of quantifying T cells reactive with HLA-matched tumorcells using established techniques such as intracellular FACS, cytokinerelease assays, and Elispot assays. Immunological assays will bestandardized by the inclusion of 1) pre-infusion PBMC and 2) an aliquotof the T cells cryopreserved at the time of infusion. TCR gene usage maybe quantitated in samples using conventional sequencing techniques ofthe T cell receptor variable regimen of the beta chain.

A variety of tests including evaluation of specific lysis and cytokinerelease, intracellular FACS of cytokine production, ELISA-spot assays,and lymphocyte subset analysis may be used to compare evaluate theimmunological correlates of treatment. In general, differences of 2 to 3fold in these assays are indicative of true biologic differences. Inaddition, measurement of CD4+ and CD8+ T cells will be conducted andstudies of cell persistence in the circulation will be conducted byusing PCR assays capable of detecting the unique sequence of the T-cellreceptor rearrangements of the infused cells.

Samples of all infused cell products will be cryopreserved, andextensive retrospective analysis of infused cell phenotype and functionwill be performed to attempt to find in vitro characteristics of theinfused cells which correlate with in vivo antitumor activity. Analysesof TIL samples will include evaluation of the activity, specificity, andtelomere length of the infused TIL.

Blood and tissue specimens collected in the course of this researchproject may be banked and used in the future to investigate newscientific questions related to this study. However, this research mayonly be done if the risks of the new questions were covered in theconsent document. If new risks are associated with the research (e.g.,analysis of germ line genetic mutations) the principal investigator mustamend the protocol and obtain informed consent from all researchsubjects.

10 cc serum and separated lymphocytes from blood will be obtained priorto cell infusion and following each infusion if possible andcryopreserved for subsequent testing; leukapheresis will be utilized toobtain peripheral blood lymphocytes in patients, pretreatment and may berepeated at approximately 4-6 weeks after the cell infusions and willconsist of a 7.5-liter exchange to last approximately three hours. Allpatients undergoing pheresis will sign informed consent.

3.4 Post Treatment Evaluation (Follow-Up) 4-6 Weeks Following CellInfusion

Patients will be evaluated on the basis of a physical examination,toxicity assessment, and CT of the chest, abdomen and pelvis. This endof course evaluation will be used to determine tumor response. Ifclinically indicated, other scans or x-rays may be performed, e.g.,brain MRI, bone scan.

A 5 liter apheresis will be performed or 60 ml of blood will beobtained. Peripheral blood mononuclear cells will be cryopreserved sothat immunologic testing may be performed.

For patients in cohort 3 only, 15 mL of clean catch urine to be testedwill be collected approximately 1 month after TBI and 6 months afterTBI.

If the patient has SD or tumor shrinkage, repeat complete evaluationswill be performed every 1-3 months, or as clinically indicated includingobtaining 60 ml of blood for immunologic testing.

4.0 Supportive Care

Concomitant medications to control side-effects of therapy will begiven. Meperidine (25-50 mg) will be given intravenously if severechilling develops. Other supportive therapy will be given as requiredand may include acetaminophen (650 mg q4h), indomethacin (50-75 mg q6h)and ranitidine (150 mg q 12h). Patients who require transfusions willreceive irradiated blood products. Additional antiemetic therapy will beadministered for breakthrough nausea and vomiting.

5.0 Data Collection and Evaluation 5.1 Response Criteria 5.1.2.Evaluation of Target Lesions

All measurable lesions up to a maximum of 10 lesions representative ofall involved organs should be identified as target lesions and recordedand measured at baseline. Target lesions should be selected on the basisof their size (lesions with the longest diameter) and their suitabilityfor accurate repetitive measurements (either by imaging techniques orclinically). A sum of the longest diameter (LD) for all target lesionswill be calculated and reported as the baseline sum LD. The baseline sumLD will be used as reference to further characterize the objective tumorresponse of the measurable dimension of the disease.

Responses are determined as set forth in Table 14. A Complete Response(CR) is considered to be the disappearance of all target lesions. APartial Response (PR) is considered to be at least a 30% decrease in thesum of the longest diameter (LD) of target lesions taking as referencethe baseline sum LD. Progression (PD) is considered to be at least a 20%increase in the sum of LD of target lesions taking as reference thesmallest sum LD recorded since the treatment started or the appearanceof one or more new lesions. Stable Disease (SD) is considered to beeither sufficient shrinkage to qualify for PR nor sufficient increase toqualify for PD taking as references the smallest sum LD.

5.1.2. Evaluation of Non-Target Lesions

All other lesions (or sites of disease) should be identified asnon-target lesions and should also be recorded at baseline. Measurementsare not required, and these lesions should be followed as “present” or“absent.”

Responses are determined as set forth in Table 14. Complete Response(CR) is considered to be the disappearance of all non-target lesions andnormalization of tumor marker level. Non-Complete Response is consideredto be the persistence of one or more non-target lesions. Progression(PD) is considered to be the appearance of one or more new lesionsand/or unequivocal progression of existing non-target lesions.

5.1.3 Evaluation of Best Overall Response

The best overall response is the best response recorded from the startof the treatment until disease progression/recurrence (taking asreference for progressive disease the smallest measurements recordedsince the treatment started). The patient's best response assignmentwill depend on the achievement of both measurement and confirmationcriteria.

TABLE 14 Target Lesions Non-Target Lesions New Lesions Overall ResponseCR CR No CR CR Non-CR/Non-PD No PR PR Non-PD No PR SD Non-PD No SD PDAny Yes or No PD Any PD Yes or No PD Any Any Yes PD

5.1.4 Confirmatory Measurement/Duration of Response Confirmation

To be assigned a status of PR or CR, changes in tumor measurements mustbe confirmed by repeat studies that should be performed at least 4 weeksafter the criteria for response are first met. In the case of SD,follow-up measurements must have met the SD criteria at least once afterstudy entry at a minimum interval of 6-8 weeks.

Duration of Overall Response

The duration of overall response is measured from the time measurementcriteria are met for CR/PR (whichever is first recorded) until the firstdate that recurrent or progressive disease is objectively documented(taking as reference for progressive disease the smallest measurementsrecorded since the treatment started).

The duration of overall complete response is measured from the timemeasurement criteria are first met for CR until the first date thatrecurrent disease is objectively documented.

Duration of Stable Disease

Stable disease is measured from the start of the treatment until thecriteria for progression are met, taking as reference the smallestmeasurements recorded since the treatment started.

Pharmaceutical Information Interleukin-2 (Aldesleukin, Proleukin,Recombinant Human Interleukin 2)

How Supplied:

Interleukin-2 (aldesleukin) is manufactured by the Chiron Corporation,Emeryville, Calif., and will be purchased by the NIH Clinical PharmacyDepartment from commercial sources.

Formulation/Reconstitution:

Aldesleukin, NSC #373364, is provided as single-use vials containing 22million IU (−1.3 mg) IL-2 as a sterile, white to off-white lyophilizedcake plus 50 mg mannitol and 0.18 mg sodium dodecyl sulfate, butteredwith approximately 0.17 mg monobasic and 0.89 mg dibasic sodiumphosphate to a pH of 7.5 (range 7.2 to 7.8). The vial is reconstitutedwith 1.2 mL of Sterile Water for Injection, USP, and the resultantconcentration is 18 million IU/ml or 1.1 mg/mL. Diluent should bedirected against the side of the vial to avoid excess foaming. Swirlcontents gently until completely dissolved. Do not shake. Since vialscontain no preservative, reconstituted solution should be used with 24hours.

Storage:

Intact vials are stored in the refrigerator (2°-8° C.) protected fromlight. Each vial bears an expiration date.

Dilution/Stability:

Reconstituted aldesleukin should be further diluted with 50 mL of 5%Human Serum Albumin (HSA). The HSA should be added to the diluent priorto the addition of RIL-2. Dilutions of the reconstituted solution over a1000-fold range (i.e., 1 mg/mL to 1 mcg/mL) are acceptable in eitherglass bottles or polyvinyl chloride bags. Aldesleukin is chemicallystable for 48 hours at refrigerated and room temperatures, 2°-30° C.

Administration:

The dosage will be calculated based on total body weight. The finaldilution of aldesleukin will be infused over 15 minutes. Aldesleukinwill be administered as an inpatient.

Toxicities:

Expected toxicities of aldesleukin are listed in the product label.Grade III toxicities common to aldesleukin include diarrhea, nausea,vomiting, hypotension, skin changes, anorexia, mucositis, dysphagia, orconstitutional symptoms and laboratory changes.

8.2 Fludarabine

Description:

Fludarabine phosphate is a synthetic purine nucleoside that differs fromphysiologic nucleosides in that the sugar moiety is arabinose instead ofribose or deoxyribose. Fludarabine is a purine antagonistantimetabolite.

How Supplied:

It will be purchased by the NIH Clinical Pharmacy Department fromcommercial sources. Fludarabine is supplied in a 50 mg vial as afludarabine phosphate powder in the form of a white, lyophilized solidcake.

Stability:

Following reconstitution with 2 mL of sterile water for injection to aconcentration of 25 mg/ml, the solution has a pH of 7.7. The fludarabinepowder is stable for at least 18 months at 2-8° C.; when reconstituted,fludarabine is stable for at least 16 days at room temperature. Becauseno preservative is present, reconstituted fludarabine will typically beadministered within 8 hours. Specialized references should be consultedfor specific compatibility information. Fludarabine is dephosphorylatedin serum, transported intracellularly and converted to the nucleotidefludarabine triphosphate; this 2-fluoro-ara-ATP molecule is thought tobe required for the drug's cytotoxic effects. Fludarabine inhibits DNApolymerase, ribonucleotide reductase, DNA primase, and may interferewith chain elongation, and RNA and protein synthesis.

Storage:

Intact vials should be stored refrigerated (2-8° C.).

Administration:

Fludarabine is administered as an IV infusion in 100 ml 0.9% sodiumchloride, USP over 15 to 30 minutes. The doses will be based on bodysurface area (BSA). If patient is obese (BMI >35) drug dosage will becalculated using practical weight.

Toxicities:

At doses of 25 mg/m²/day for 5 days, the primary side effect ismyelosuppression; however, thrombocytopenia is responsible for mostcases of severe and life-threatening hematologic toxicity. Seriousopportunistic infections have occurred in CLL patients treated withfludarabine. Hemolytic anemia has been reported after one or morecourses of fludarabine with or without a prior history of a positiveCoomb's test; fatal hemolytic anemia has been reported. In addition,bone marrow fibrosis has been observed after fludarabine therapy. Othercommon adverse effects include malaise, fever, chills, fatigue,anorexia, nausea and vomiting, and weakness. Irreversible andpotentially fatal central nervous system toxicity in the form ofprogressive encephalopathy, blindness, and coma is only rarely observedat the currently administered doses of fludarabine. More commonneurologic side effects at the current doses of fludarabine includeweakness, pain, malaise, fatigue, paresthesia, visual or hearingdisturbances, and sleep disorders. Adverse respiratory effects offludarabine include cough, dyspnea, allergic or idiopathic interstitialpneumonitis. Tumor lysis syndrome has been rarely observed infludarabine treatment of CLL. Treatment on previous adoptive celltherapy protocols in the Surgery Branch have caused persistently low(below 200) CD4 counts, and one patient developed polyneuropathymanifested by vision blindness, and motor and sensory defects.

Cyclophosphamide

Description:

Cyclophosphamide is a nitrogen mustard-derivative alkylating agent.Following conversion to active metabolites in the liver,cyclophosphamide functions as an alkyating agent; the drug alsopossesses potent immunosuppressive activity. The serum half-life afterIV administration ranges from 3-12 hours; the drug and/or itsmetabolites can be detected in the serum for up to 72 hours afteradministration.

How Supplied:

Cyclophosphamide will be obtained from commercially available sources bythe Clinical Center Pharmacy Department.

Stability:

Following reconstitution as directed with sterile water for injection,cyclophosphamide is stable for 24 hours at room temperature or 6 dayswhen kept at 2-8° C.

Administration:

It will be diluted in 250 ml D5W and infused over one hour. The dosewill be based on the patient's body weight. If patient is obese(BMI >35) drug dosage will be calculated using practical weight.

Toxicities:

Hematologic toxicity occurring with cyclophosphamide usually includesleukopenia and thrombocytopenia. Anorexia, nausea and vomiting, rash andalopecia occur, especially after high-dose cyclophosphamide; diarrhea,hemorrhagic colitis, infertility, and mucosal and oral ulceration havebeen reported. Sterile hemorrhagic cystitis occurs in about 20% ofpatients; severity can range from microscopic hematuria to extensivecystitis with bladder fibrosis. Although the incidence of hemorrhagiccystitis associated with cyclophosphamide appears to be lower than thatassociated with ifosfamide, mesna (sodium 2-mercaptoethanesulfonate) hasbeen used prophylactically as a uroprotective agent in patientsreceiving cyclophosphamide. Prophylactic mesna is not effective inpreventing hemorrhagic cystitis in all patients. Patients who receivehigh dose cyclophosphamide may develop interstitial pulmonary fibrosis,which can be fatal. Hyperuricemia due to rapid cellular destruction mayoccur, particularly in patients with hematologic malignancy.Hyperuricemia may be minimized by adequate hydration, alkalinization ofthe urine, and/or administration of allopurinol. If allopurinol isadministered, patients should be watched closely for cyclophosphamidetoxicity (due to allopurinol induction of hepatic microsomal enzymes).At high doses, cyclophosphamide can result in a syndrome ofinappropriate antidiuretic hormone secretion; hyponatremia withprogressive weight gain without edema occurs. At high doses,cyclophosphamide can result in cardiotoxicity. Deaths have occurred fromdiffuse hemorrhagic myocardial necrosis and from a syndrome of acutemyopericarditis; in such cases, congestive heart failure may occurwithin a few days of the first dose. Other consequences ofcyclophosphamide cardiotoxicity include arrhythmias, potentiallyirreversible cardiomyopathy, and pericarditis. Other reported adverseeffects of cyclophosphamide include headache, dizziness, and myxedema;faintness, facial flushing, and diaphoresis have occurred following IVadministration. Mesna (sodium 2-mercaptoethanesulphonate; given by IVinjection) is a synthetic sulfhydryl compound that can chemicallyinteract with urotoxic metabolites of cyclophosphamide (acrolein and4-hydroxycyclophosphamide) to decrease the incidence and severity ofhemorrhagic cystitis.

Mesna (Sodium 2-mercaptoethanesulfonate, Mesnum, Mesnex, NSC-113891)

Description:

Mesna will be obtained commercially by the Clinical Center PharmacyDepartment and is supplied as a 100 mg/ml solution.

Storage:

Intact ampoules are stored at room temperature.

Stability:

Diluted solutions (1 to 20 mg/mL) are physically and chemically stablefor at least 24 hours under refrigeration. Mesna is chemically stable atroom temperature for 48-72 hours in D5W, 48-72 hour in D5W/0.45% NaCl,or 24 hours in 0.9% NaCl.

Administration:

Dilute to concentrations less than or equal to 20 mg mesna/ml fluid inD5W or 0.9% NaCl and to be administered intravenously as a continuousinfusion. If patient is obese (BMI >35) drug dosage will be calculatedusing practical weight. Toxicities include nausea, vomiting anddiarrhea.

Filgrastim (Granulocyte Colony-Stimulating Factor, G-CSF, Filgrastim,Neupogen)

Filgrastim will be obtained commercially by the Clinical Center PharmacyDepartment and is supplied in 300 ug/ml and 480 ug/1.6 ml vials. G-CSFshould be refrigerated and not allowed to freeze. The product bears theexpiration date. The product should not be shaken. It is generallystable for at least 10 months when refrigerated. The appropriate dose isdrawn up into a syringe. G-CSF will be given as a daily subcutaneousinjection. The side effects of G-CSF are skin rash, myalgia and bonepain, an increase of preexisting inflammatory conditions, enlargedspleen with occasional associated low platelet counts, alopecia (withprolonged use) elevated blood chemistry levels.

Trimethoprim and Sulfamethoxazole Double Strength (TMP/SMX DS)

TMP/SMX DS will be obtained by the Clinical Center Pharmacy Departmentfrom commercial sources. It will be used for the prevention of PCPpneumonia. The oral dose is 1 tablet PO daily three times a week (onNON-consecutive days) beginning on day −7 and continuing for at least 6months and until the CD4 count is greater than 200 on 2 consecutive labstudies. Like other sulfa drugs, TMP/SMX DS can cause allergies, fever,photosensitivity, nausea, and vomiting. Allergies typically develop as awidespread itchy red rash with fever eight to fourteen days afterbeginning the standard dose. Neutropenia, a reduction in the number ofneutrophils, can also occur.

Aerosolized Pentamidine in Place of TMP/SMX DS:

Patients with sulfa allergies will receive aerosolized Pentamidine 300mg per nebulizer within one week prior to admission and continuedmonthly until the CD4 count is above 200 on two consecutive follow uplab studies and for at least 6 months post chemotherapy. PentamidineIsethionate will be obtained by the Clinical Center Pharmacy Departmentfrom commercial sources. It will be used to prevent the occurrence ofPCP infections. It is supplied in 300 mg vials of lyophilized powder andwill be administered via nebulizer. Toxicities reported with the use ofPentamidine include metallic taste, coughing, bronchospasm in heavysmokers and asthmatics; increased incidence of spontaneous pneumothoraxin patients with previous PCP infection or pneumatoceles, orhypoglycemia.

Herpes Virus Prophylaxis Valacyclovir (Valtrex)

Valacyclovir will be obtained by the Clinical Center Pharmacy Departmentfrom commercial sources. It will be used orally to prevent theoccurrence of herpes virus infections in patients with positive HSVserology. It is supplied in 500 mg tablets. Valacyclovir will be startedthe day after the last dose of fludarabine at a dose of 500 mg orallydaily if the patient is able to tolerate oral intake. See package insertfor dosing adjustments in patients with renal impairment. Common sideeffects include headache, upset stomach, nausea, vomiting, diarrhea orconstipation. Rare serious side effects include hemolytic uremicsyndrome and thrombotic thrombocytopenic purpura.

Acyclovir

Acyclovir will be obtained by the Clinical Center Pharmacy Departmentfrom commercial sources. It will be used to prevent the occurrence ofherpes virus infections in patients who cannot take oral medications. Itis supplied as powder for injection in 500 mg/vials. Reconstitute in 10mL of sterile water for injection to a concentration of 50 mg/mL.Reconstituted solutions should be used within 12 hours. IV solutionsshould be diluted to a concentration of 7 mg/mL or less and infused over1 hour to avoid renal damage. Reversible renal insufficiency has beenreported with IV but not oral acyclovir. Neurologic toxicity includingdelirium, tremors, coma, acute psychiatric disturbances, and abnormalEEGs have been reported with higher doses of acyclovir. Should thisoccur, a dosage adjustment will be made or the drug will bediscontinued. Stomach upset, headache or nausea, rash or hives;peripheral edema; pain, elevated liver function tests; and leukopenia,diarrhea, lymphadenopathy, myalgias, visual abnormalities and elevatedcreatinine have been reported. Hair loss from prolonged use has beenreported. Acyclovir will not be used concomitantly with other nucleosideanalogs which interfere with DNA synthesis, e.g. ganciclovir. In renaldisease, the dose is adjusted as per product labeling.

Fluconazole

Fluconazole will be obtained by the Clinical Center Pharmacy Departmentfrom commercial sources. It will be used to prophylax against fungalinfections. It is available in 200 mg tablets. It can cause headache,nausea, vomiting, diarrhea or abdominal pain, and liver damage which maybe irreversible. It can cause rashes and itching, which in rare caseshas caused Stevens Johnson Syndrome. It has several significant druginteractions. The package insert should be consulted prior toprescribing. For IV administration in patients who cannot tolerate theoral preparation, Fluconazole comes in 2 MG/ML solution for injection,and prepared according to Clinical Center Pharmacy standard procedures.It should be administered at a maximum IV rate of 200 mg/hr.

OKT3

OKT3 will be obtained by the Surgery Branch Laboratory from commercialsources.

Formulation:

Muromonab-CD3 (Ortho), NSC #618843, is provided as a sterile, clear,colorless solution at a concentration of 1 mg/ml in 5 ml ampoules. Thesolution may contain a few fine, translucent protein particles. Theantibody is dissolved in a buffered solution at pH of 6.5 to 7.5. Thesolution contains 2.25 mg of monobasic sodium phosphate, 9 mg of dibasicsodium phosphate, 43 mg of sodium chloride and 1 mg of polysorbate 80per 5 ml of water for injection.

Storage/Stability:

Ampules should be stored in a refrigerator at 2-8° C. Solution shouldnot be frozen or shaken. Each ampule bears an expiration date.

Support Medications Ondansetron Hydrochloride

Ondansetron hydrochloride will be obtained by the Clinical CenterPharmacy Department from commercial sources. It will be used to controlnausea and vomiting during the chemotherapy preparative regimen. It cancause headache, dizziness, myalgias, drowsiness, malaise, and weakness.Less common side effects include chest pain, hypotension, pruritis,constipation and urinary retention. Consult the package insert forspecific dosing instructions.

Furosemide

Furosemide will be obtained by the Clinical Center Pharmacy Departmentfrom commercial sources. It will be used to enhance urine output duringthe chemotherapy preparative regimen with cyclophosphamide. Adverseeffects include dizziness, vertigo, paresthesias, weakness, orthostatichypotension, photosensitivity, rash and pruritis. Consult the packageinsert for a complete list of all side effects.

Preliminary Results

Twenty-five patients underwent treatment including administration ofyoung TIL that were rapidly expanded and administered (ages of young TILin days: 20, 24, 26, 26, 26, 26, 27, 27, 28, 28, 29, 29, 29, 29, 32, 34,34, 34, 34, 34, 35, 35, 35, 36, 36) following lymphodepletion withcyclophosphamide and fludarabine. Patients then received high doseinterleukin (IL)-2 therapy. Six patients (24%) experienced an objectiveresponse to young TIL therapy. There were significant toxicitiesassociated with TIL administration including seven instances of adverseevents requiring intubation and one treatment related mortality.Subsequently permission was sought and obtained to amend the protocol toenrich the TIL population for CD8⁺ cells prior to rapid expansion, asset forth in Example 12.

This example demonstrated a method of promoting regression of a cancerin a mammal comprising (i) culturing autologous T cells; (ii) rapidlyexpanding the cultured T cells; (iii) administering to the mammalnonmyeloablative lymphodepleting chemotherapy; and (iv) afteradministering nonmyeloablative lymphodepleting chemotherapy,administering to the mammal the expanded T cells, wherein the T cellsadministered to the mammal are about 19 to about 35 days old, whereuponthe regression of the cancer in the mammal is promoted.

Example 12

This example demonstrates the generation of CD8+ enriched young TIL.Patients with metastatic melanoma underwent biopsy and as much of thesample as possible was processed to a single cell suspension forgeneration of young TIL as described in Example 1, Dudley et al. J.Immunother. 26(4):332-42 (2003) and as further detailed in this example.

Acquisition of Samples for Initiation of TIL.

The specimen is received in the laboratory as soon as possible aftersurgery. All appropriate procurement documentation must accompanyclinical specimens. Once the specimen arrives in the laboratory, alldownstream processing is performed in a laminar flow biological safetycabinet. The sample is assigned a unique tumor accession number. With aclinical pathologist present, tissue is dissected that will remain inthe Cell Production Laboratory free from normal, necrotic, and excesssample, and the latter is returned to the pathology laboratory fordiagnosis. Additional research samples are taken for cytopathology,immunocytochemistry, and RNA analysis as necessary. The remaining tumorsample is now ready to prepare as a single cell suspensions.

Preparation of Single Cell Suspension.

Samples smaller than 5 g may be prepared by physical disaggregation inenzymes media using a gentleMACS™ Dissociator (Miltenyi Biotec, Auburn,Calif.) according to the manufacturers recommendations (Miltenyi).Samples larger than 5 g may be prepared by overnight enzymaticdissociation. Tissue should be diced into approximately 2 mm pieces andincubated in enzyme media with gentle stirring overnight. Enzyme mediaconsists of RPMI 1640 (without serum) containing penicillin G (100units/ml), streptomycin (100 μg/ml), gentamicin (50 μg/ml), Fungizone(1.25 μg/ml) (Bristol-Myers Squibb Co.; Princeton, N.J.), Collagenase(Type IV, Sigma, 1 mg/ml) and Pulmozyme® (Dornase, Genentech, SanFrancisco, Calif. ˜30 units/ml). Disaggregated tumor samples should bepassed through a 100 uM wire mesh or 70 uM disposable strainer, andwashed three times prior to counting and plating. The single cellsuspension was evaluated on a hemacytometer with lymphocytes and tumorcells determined based on size and morphology and viability determinedby trypan blue staining. Cell composition is evaluated usingmorphological criteria to distinguish erythrocytes, lymphocytes andtumor cells. Tumor digest preparations that contain more than 50% deadcells or more than 80% erythrocytes may be further processed byFicoll-hypaque step gradient enrichment.

Establishment of “bulk” TIL Cultures

TIL cultures are set up as described in Example 1. Briefly, cells areplated in 24-well sterile tissue culture plates, using 5.0×10⁵ totalviable nucleated cells/ml (1.0×10⁶ total viable cells/well, 2 ml/well)in complete medium (CM) containing 6000 IU/ml IL-2. The plates areincubated in a humidified incubator at 37° C., with 5% CO₂ in air. CMconsists of RPMI-1640 with 10% human serum plus penicillin G (100units/ml), streptomycin (100 μg/ml), gentamicin (50 μg/ml), andL-glutamine (146 μg/ml, 1 mM). The antibiotics may be omitted if apatient has relevant allergies. Other GMP quality media additives may beused when necessary (e.g. imipenem for potentially contaminated bowellesions). Starting 5 days to one week after set up, half of the CM isreplaced with fresh medium containing 6000 IU/ml IL-2. 2-3 media changesshould be performed per week until the culture exceeds 1.0×10⁶lymphocytes/ml or becomes nearly confluent. As soon as TIL growth in thewells becomes confluent (and all adherent cells are eliminated), thenindividual culture wells should be pooled. Aliquots may be cryopreservedor the bulk TIL cells may be expanded further by re-plating at0.7−1.5×10⁶ lymphocytes/ml in CM containing IL-2 until sufficient cellsare obtained for therapy. An aliquot of the bulk TIL should be examinedby FACS to determine the percentages of CD3⁺ and CD8⁺ cells. The resultsare shown in Table 15.

CD8+ Enrichment and Clinical Scale Rapid Expansion Protocol (REP).

After minimum time in culture, successfully initiated “bulk” young TILwere CD8+ enriched (Miltenyi CliniMACS) (Prieto et al. J. Immunother.33(5):547-556 (2010)) and rapidly expanded to clinical cell numbers(Dudley et al. J. Clin. Oncol. 23(10):2346-57 (2005); Riddell et al. J.Immunol. Methods 128(2):189-201 (1990)). The CD8+ enrichment has beendescribed in detail (Prieto et al. J. Immunother. 33(5):547-556 (2010))and is a modification of the Miltenyi CliniMACS procedure recommended bythe manufacturer.

The rapid expansion protocol (REP) was modified from Riddell et al. J.Immunol. Methods 128(2):189-201 (1990) and has been described previously(Dudley et al. J. Clin. Oncol. 23(10):2346-57 (2005)). Briefly, 1×10⁶CD8+ enriched “responder” TIL are mixed with a 1:200 excess of 40Gyirradiated peripheral blood mononuclear “feeder” cells in 150 ml of “50/50” media containing 3000 IU/ml IL-2 and 30 ng/ml OKT3(Ortho-McNeil®, Raritan, N.J.). 50/50 media consists of a 1:1 mixture ofCM and AIM V (Invitrogen, Carlsbad Calif.). The mixture is added to aT175 flask and incubated in a vertical position in a humidifiedincubator at 37° C. in 5% CO₂ atmosphere. After 5 days, approximately ⅔of the media is replaced with fresh 50/50 media containing IL-2 with noOKT3. After day 7, cells are maintained by splitting with AIM V mediasupplemented with 3000 IU/ml IL-2 as needed to maintain cell densitiesaround 1×10⁶ cells/ml. Cells are harvested by continuous flowcentrifugation, washed, and infused on day 14.

Aliquots of infused samples were evaluated by FACS and cytokine releaseassays to determine lymphocyte phenotype and antigen specificityrespectively using standard techniques (Dudley et al. J. Clin. Oncol.23(10):2346-57 (2005)). The results are shown in Table 15. Table 15 setsforth the phenotype of bulk TIL cultures prior to CD8+ enrichment (TILpre separation) and CD8+ enriched TIL products infused for therapy(Infusion Bag). “Cells” indicates the number of cells infused fortreatment. Other numbers indicate the percent of total cells in thepopulation expressing each marker (“NK” indicates CD56+ CD3− phenotype).Blank entries indicate data not obtained. NK cells (CD56+ CD3−) were notdetermined for patients who received NMA conditioning.

TABLE 15 NMA Treatment 6Gy TBI Treatment Cells TIL Pre SeparationInfusion Bag Cells TIL Pre Separation Infusion Bag (x10e9) CD4 CD8 NKCD4 CD8 (x10e9) CD4 CD8 NK CD4 CD8 NK 84.6 6 69 10 0 98 22.5 4 46 35 197 0 72.7 11 39 9 0 98 32.8 26 54 16 1 100 0 56.9 11.8 12 57 11 0 99 024.5 32 44 19 5 97 20.1 1 99 0 23.8 2 97 41.2 49 25 22 2 97 0 34.2 19 8511 1 98 54.3 19 61 12 1 99 0 36.2 53 16 37 2 99 48.3 48 11 30 3 98 047.6 5 81 37 0 99 39.2 18 29 53 0 98 0 41.4 38 53 8 0 99 35.9 14 30 44 198 0 55.9 22 80 3 0 100 46.5 38 17 36 19 85 0 84.3 32 56 27 0 88 51.7 582 9 0 99 0 55.1 25 67 18 1 95 44.7 4 88 5 6 84 1 41.3 35 42 4 1 95 32.536 37 16 1 98 0 50.3 30 59 20 1 91 33.7 40 39 13 2 99 0 51.1 3 96 3 0 9635.8 3 66 37 0 99 0 65.2 3 91 14 0 99 29.1 16 50 8 2 98 0 76.4 4 91 3 099 71.0 41 56 3 0 100 0 67.8 32 62 34 0 86 57.6 8 77 7 0 100 0 44.6 1480 5 1 98 97.5 65 32 1 2 99 0 59.1 28 42 29 1 97 74.9 17 79 2 0 99 025.1 19 32 36 0 95 35.4 14 24 36 0 99 0 21.2 56 24 29 1 97 31.4 16 36 430 96 0 67.8 18 62 18 0 94 43.1 6 32 4 1 99 0 42.9 17 75 7 0 98 Average43.1 22.7 49.0 20.1 2.1 97.3 0.1 61.0 7 52 34 0 96 St. 4.0 3.8 4.9 3.40.9 0.9 0.0 50.6 23 76 5 0 92 Error 27.2 19 45 33 0 98 43.9 9 47 31 0 9730.8 4 50 41 0 99 31.0 26 12 53 0 98 44.8 1 55 46 0 96 5.8 80 8 3 8 8847.6 6 81 19 Average 47.7 21.8 57.2 20.8 0.8 96.0 St. 3.3 3.2 4.3 2.60.3 0.6 Error

The CD8+ enrichment was highly effective for reducing the fraction ofCD4+ cells in the infused TIL, as shown in Table 15. The CD4+ cellcomponent comprised an average of 22% of the cells in bulk young TILprior to CD8+ enrichment, and NK cells comprised 21% (Table 15). TILfrom prior protocols administered without CD8+ enrichment after NMAconditioning also contained about 21% CD4+ cells. Following CD8+enrichment and expansion, CD4+ cells were reduced to an average of 2% ofthe infused young TIL.

This example demonstrated a method of generating CD8+ enriched, “young”T cells, and also demonstrated that the CD8+ enrichment was highlyeffective for reducing the fraction of CD4+ cells in the infused TIL.

Example 13

This example demonstrates that the administration of CD8+ enriched,“young” T cells promotes the regression of cancer in human melanomapatients.

Patients, Clinical Samples and Trial Design

Patients were eligible for this study who were 18 years or older withmeasurable metastatic melanoma, at least one lesion respectable for TIL,good clinical performance, adequate liver and kidney function tests,blood counts near the normal range, free from active systemic infectionswithout coagulation disorders or cardiovascular disease orimmunodeficiency, negative for HIV antibody and hepatitis B and C, and alife expectancy of greater than three months. All patients signed aninformed consent approved by the Institutional Review Board of theNational Cancer Institute.

One group of 33 patients received non-myeloablative chemotherapy (NMA)consisting of 60 mg/kg/day cyclophosphamide for two days followed byfive days of 25 mg/m²/day fludarabine. A second cohort of 23 patientsreceived two days of 60 mg/kg cyclophosphamide overlapping the first twoof five days of 25 mg/m²/day fludarabine. On the final day offludarabine, patients received two fractions of 2Gy total bodyirradiation (TBI) separated by at least 6 hrs, and the following daythey received one fraction of 2Gy TBI. On the day following chemotherapyor radiation all patients received a bolus intravenous infusion of CD8+enriched young TIL (prepared generally as described in Example 12) andstarted high dose IL-2 therapy (720,000 IU/kg intravenously every 8 hrsto tolerance). The age (in days) of the CD8+ enriched young TILadministered to patients receiving NMA is as follows: 26, 27, 27, 28,28, 29, 29, 30, 30, 30, 31, 31, 31, 31, 31, 32, 32, 34, 34, 34, 35, 35,35, 35, 35, 36, 36, 37, 37, 41, 41, and 41 (average age is given inTable 16). The age (in days) of the CD8+ enriched young TIL administeredto patients receiving TBI is as follows: 28, 29, 29, 30, 30, 30, 30, 31,33, 33, 34, 35, 35, 35, 37, 37, 38, 38, 39, 42, 43, 47, and 53 (averageage is given in Table 16).

One day after TIL infusion, patients who received 6Gy TBI received aminimum of 2×10⁶/kg autologous purified (Miltenyi) CD34+ hematopoieticstem cells from a G-CSF+ plerixafor mobilized pheresis.

Patients received trimethoprim, sulfamethoxazole, and fungal prophylaxisfollowing therapy; herpes virus seropositive patients also receivedvalacyclovir. Platelets and packed red blood cells were administered asneeded during hematopoietic recovery, and empiric antibiotics wereinitiated for neutropenic fevers (38.3° C. once or two temperatures of38.0° C. at least one hour apart and absolute neutrophil count <500).Patient response was assessed using standard radiographic studies andphysical examination at approximately four weeks following TILadministration and at regular intervals thereafter. The ResponseEvaluation Criteria In Solid Tumors (RECIST) guidelines were followedand patients were categorized into complete, partial, or non-respondingcategories. Complete blood counts (CBC) were obtained at least once perday while patients were in the hospital and differential counts wereobtained when CBC was over 200 cells per microliter.

The demographic characteristics of these patients and the treatmentsadministered are shown in Table 16.64% of patients had received priorIL-2. Median follow up in the NMA and 6Gy TBI cohorts was 14 months and7 months, respectively.

TABLE 16 NMA 6Gy TBI Patients 33 23 Sex Male 14 13 Female 19 10 Age(years) ≦30 3 3 30-39 6 5 40-49 12 4 50-59 10 11 ≧60 2 0 HLA-A2+ Yes 1110 No 22 13 Prior IL-2 Yes 25 11 No 8 12 Stage of disease M1a 2 1 M1b 1110 M1c 20 12 *Cell number (×10⁹) 47.7 (±3.3) 43.1 (±7.5) IL-2 (doses) 6.3 (±0.3)  7.5 (±0.5) Average Age of cells at 32.7 (±0.7) 35.4 (±1.3)Infusion (days) CD4+ cells (%) Prior to CD8+ enrichment 21.8 (±3.2) 22.7(±3.6) Infused  0.6 (±0.3)  2.1 (±0.8) CD8+ cells (%) Prior to CD8+enrichment 57.2 (±4.3) 49.0 (±4.7) Infused 96.0 (±0.6) 97.3 (±0.8)Tissue of TIL origin^(†) Lymph node 16 6 Subcutaneous 10 7 Liver 4 3Lung 3 5 Large bowel 0 1 Intramuscular 0 2 Other visceral site 2 1*Average (±Standard Error) ^(†)Some patients were treated with TIL frommultiple tissues of origin

Nineteen of the 33 patients (58%) in the NMA cohort exhibited anobjective tumor regression by RECIST criteria, including 16 partialresponders (48%), and 3 complete responders (9%). Eleven of 23 patients(48%) in the 6Gy TBI cohort achieved an objective response, includingtwo complete responders (9%).

Illustrative examples of clinical tumor regression are shown in FIGS.4A, 4B, and 4C. CD8+ enriched young TIL caused regression of bulkymelanoma lesions at multiple sites. FIG. 4A shows CT scans of metastaticmelanoma lesions (arrows) in a first patient before CD8+ enriched youngTIL therapy in the sacrum and ilium (top left), lung (middle left) andspleen (lower left). As shown in FIG. 4A, all lesions showed regressionat two months (right) with visible signs of recalcification of priormetastatic sites in the sacrum and ilium. FIG. 4B (left panel) is aphotograph showing subcutaneous melanoma around the ear and in theauditory canal that caused complete hearing loss in a second patient.FIG. 4B (middle panel) is a photograph showing that eleven days afterCD8+ enriched young TIL infusion, gross necrosis of the melanoma wasvisible. FIG. 4B (right panel) is a photograph showing that at 76 daysafter treatment, the patient experienced a partial response at all sitesincluding liver and subcutaneous lesions. Tumor was absent from theauditory canal and the patient's hearing returned to normal. FIG. 4Cshows CT scans illustrating mediastinal, lung, nodal and subcutaneousmetastatic deposits in a third patient before (Left) and one month after(Right) treatment with CD8+ enriched young TIL, demonstrating the rapidinitial pace of tumor regression in a patient who eventually achieved acomplete response.

Fifteen of 24 patients with M1a or M1b melanoma and 15 out of 32patients with M1c disease responded to therapy. As reported previously(Dudley et al. J. Clin. Oncol. 26(32):5233-9 (2008); Dudley et al. J.Clin. Oncol. 23(10):2346-57 (2005)) all patients experienced transienthematological toxicities from the lymphodepleting conditioning andreceived platelet and red blood cell transfusions as medicallyindicated. Patients were also treated for symptoms associated with highdose IL-2 therapy. All toxicities typically returned to baseline withina few days. All non-hematological grade 3 and 4 toxicities notattributable to IL-2 are listed in Table 17. There were no Grade 3 or 4toxicities directly attributable to the infused cells. There were twotreatment related mortalities, one in each cohort that resulted fromacute sepsis during the neutropenic period associated withlymphodepletion about five days after TIL infusion.

TABLE 17 NMA 6Gy TBI Total Total Patients 33  23  56 Clinical Responses(RECIST) Non-response 14 (42%) 12 (52%) 26 (46%) Partial Response 16(48%)  9 (39%) 25 (45%) Durations (months) 16+, 15+, 14+, 9+, 8+, 6, 5+,14, 13+, 13, 12, 4+, 4+, 4, 3, 2 10, 9, 8, 8, 7, 6, 5, 3, 2 CompleteResponse 3 (9%) 2 (9%) 5 (9%) Durations (months) 18+, 15+, 12+ 5+, 5+Toxicities^(†) Positive blood culture 8 4 12 (21%) Febrile neutropenia17  11  28 (50%) (Grade 3) Intubation 2 2 4 (7%) Treatment related 1 1 2(4%) death ^(†)Listed once for each patient at the highest grade. UsualIL-2 related toxicities not listed.

The age of TIL was compared for patients who received CD8+ enriched TILand patients on prior TIL protocols (FIG. 5B). Prior protocols requiredall TIL to undergo individualized testing for tumor reactivity (SpecificTIL, n=92). TIL from non-responding patients (n=40, NR) spentsignificantly longer time in culture prior to administration than TILfrom objective responders (n=52, OR). There was no difference (NS)between the time spent in culture of CD8+ enriched young TIL (Young TIL)administered to non-responding patients (n=26) or responders (n=30).Microculture generated, tumor-selected TIL administered to respondingpatients were significantly younger than TIL given to patients who didnot respond. In the cohorts treated in Example 13, there was nodifference between the age of CD8+ enriched young TIL cultures forresponding and non-responding patients, but these cultures weresignificantly younger than TIL cultures administered on prior protocols(p2=3×10⁻⁷).

This example demonstrated that the administration of CD8+ enriched,“young” T cells promotes the regression of cancer in human melanomapatients.

Example 14

This example demonstrates that the generation of “young” TIL fortreatment is reliable and rapid.

176 tumors from 122 patients were processed to establish young TILcultures, as described in Example 12. TIL were successfully grown(>50×10⁶ cells within five weeks) from 124 of the 176 lesions (70%),comprising 101 of the 122 patients (83%).

Specimens from 122 sequential patients who were eligible for TIL therapywere processed to single cell suspensions. The fraction of lymphocytesamong viable cells was determined by morphological criteria after trypanblue staining. The samples were plotted in FIG. 5A based on whethersufficient TIL grew to use for treatment (>5×10⁷ cells in 28 days, RxTIL) or whether growth was insufficient for treatment (No growth). Astriking correlation was observed between the success of establishingTIL and the initial proportion of lymphocytes in the single cellsuspension (FIG. 5A). Tumors that successfully yielded TIL had aninitial median of 52% lymphocytes while tumors that failed to grow invitro had a median of 8% lymphocytes (p2=5×10⁻⁸).

Among these 122 patients, 53 patients were treated (three additionalpatients received cryopreserved TIL from prior resections), 21 patientshad samples that failed to grow TIL cultures, 20 patients developedrapidly progressive disease that prevented treatment, 13 patients wereresected free of evaluable disease (although nine patients recurred andreceived TIL treatment subsequently), nine patients received othertreatments including one complete responder to high dose IL-2 therapy,and six patients experienced individual laboratory or clinical issues,including exactly one patient whose CD8+ TIL failed to expand during therapid expansion protocol (REP).

This example demonstrated that bulk TIL were generated within five weeksprior to rapid expansion for 83% of the patients studied, and that thepercent of lymphocytes in the initial single cell suspension correlatedwith TIL growth.

Example 15

This example demonstrates that an objective response can be obtainedusing CD8+ enriched, “young” TILs that do not exhibit tumor recognitionin vitro.

The ability of the administered CD8+ enriched TIL to recognize tumor wasretrospectively evaluated, looking for any correlation with clinicalefficacy. Recognition of autologous or HLA—matched tumor was evaluatedby cytokine release assay for CD8+ enriched young TIL administered topatients after NMA conditioning (FIG. 6A) or for CD8+ enriched young TILadministered to patients after 6Gy TBI conditioning (FIG. 6B). TIL fromcryopreserved aliquots of each infused treatment was thawed and restedovernight in IL-2, then washed and incubated at a 1:1 ratio withautologous, HLA-matched, or HLA-mismatched tumors. Interferon(IFN)-gamma secreted in the coculture supernatant was quantified byELISA. The data from each separate coculture assay was aggregated andplotted (FIGS. 6A and 6B).

Twenty-three of the 33 NMA patients had autologous tumor available (18with cryopreserved tumor digest and 5 with a tumor cell line). Eight of10 non-responding patients and 8 of 13 objective responders demonstratedautologous tumor recognition. Four additional objective respondingpatients recognized HLA-A matched tumor cell lines. Nineteen of 23patients treated with 6Gy TBI had autologous tumor available (15 withfresh frozen tumor, and four with a cell line). 8 of 11 non-respondingpatients and 5 of 8 responding patients demonstrated specific tumorrecognition. In addition, three patients' TIL recognized HLA-matchedtumor cell lines, including one non-responding patient (SA) whose TILfailed to recognize autologous tumor. In total, 29 of 42 evaluable CD8+enriched young TIL samples (69%) demonstrated specific autologous tumorrecognition and 36 of 56 (64%) patients demonstrated specificrecognition at all. Strikingly, 11 of 30 objective responses weremediated by CD8+ enriched young TIL with no evidence of specific tumorrecognition as defined in prior TIL clinical protocols.

Previously, ninety-three patients were treated over 84 months usinghighly expanded TIL selected for specific tumor recognition (Dudley etal. J. Clin. Oncol. 26(32):5233-9 (2008)), corresponding to about onetreatment per month and about 27% of resected patients who finallyreceived a TIL product. As described in Example 13, 56 additionalpatients were treated with CD8+ enriched young TIL over 14 months,corresponding to about 3 to 4 patients treated per month with 53% ofeligible patients who underwent resection able to receive TIL therapy.With both methods, clinical response rates were about 55%, but 11objective responders out of 30 in the study described in Example 13 hadTIL that would have been ineligible for the prior study.

This example demonstrated that 11 of 30 objective responses weremediated by CD8+ enriched young TIL that did not demonstrate tumorreactivity in vitro.

Example 16

This example demonstrates that CD8+ enriched, “young” TIL exhibit ahigher capacity for in vivo expansion compared to selected TIL.

The average absolute lymphocyte count (ALC) for all patients whoreceived CD8+ enriched young TIL after NMA or 6Gy TBI lymphodepletionwas determined daily and is plotted in FIGS. 7A and 7B, respectively.Not all patients had ALC determined every day. For comparison,lymphocyte reconstitution from patients who received antigen selectedTIL derived from microculture expansions after NMA or 12Gy TBI (Dudleyet al. J. Clin. Oncol. 26(32):5233-9 (2008)) lymphodepletion are alsoshown. As shown in FIGS. 7A and 7B, CD8+ enriched young TIL quicklyrepopulated patient peripheral blood to high levels after NMA or 6Gy TBIconditioning. Interestingly, patients who received CD8+ enriched youngTIL demonstrated higher peak ALC's, suggesting that CD8+ enriched youngTIL have increased capacity for in vivo expansion compared to selectedTIL.

Example 17

This example demonstrates that there is an increase in CD8+ ALC inresponding patients compared to non-responders approximately one monthafter cell infusion.

CD4+ and CD8+ ALC was determined in a blinded manner by the ClinicalCenter Core Immunology Laboratory for 47 of 56 patients treated withCD8+ enriched young TIL at approximately one month after TIL infusion.PBL from 47 of the 56 patients who received CD8+ enriched young TIL weresampled at approximately one month after cell infusion. ALC and absoluteCD3+ CD8+ and CD3+ CD4+ cell numbers were assessed by FACS analysis in ablinded manner. The treatment resulted in low CD4 counts at one month inmost patients (average: 109 CD4+ cells/up, with no difference betweenresponding and non-responding patients (p2=0.5; FIG. 7D). Patients inprior clinical trials who received TIL that contained CD4+ lymphocyteshad higher peripheral CD4+ cell counts at one month after TIL infusion(average: 187 cells/ul). In contrast to CD4+ cells, CD8+ ALC for mostpatients treated with CD8+ enriched young TIL was in the normal range atone month, and there was a significant increase in CD8+ ALC inresponding patients compared to non-responders (p2=.002; FIG. 7C).Patients who responded had CD8+ ALC about two fold higher thannon-responders (1504 vs. 696 cells/up. In prior clinical trials, theaverage CD8+ cell count was 652 CD8+ cells/ul at one month after TILinfusion. Infused TIL were examined by FACS for expression of additionalmarkers including CD27, CD28, CD62L, CCR7, but none correlated withclinical response.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

1-22. (canceled)
 23. A method of generating T cells comprising (i)culturing autologous T cells; and (ii) expanding the cultured T cellsusing OKT3 antibody, IL-2, and feeder lymphocytes, wherein the culturedT cells are enriched for CD8+ T cells prior to expansion of the T cells;and producing T cells that are about 19 to about 35 days old.
 24. Themethod according to claim 23, comprising producing T cells having a) ahigher expression of CD27 and/or CD28 than T cells that are about 44days old; b) a mean telomere length that is longer than that of T cellsthat are about 44 days old; and/or c) a higher frequency of CD4+ cellsthan T cells that are about 44 days old.
 25. The method of claim 23,wherein the T cells have not been screened for specific tumorreactivity.
 26. The method according to claim 23, comprising modifyingthe T-cells to express a T-cell growth factor that promotes the growthand activation of the autologous T-cells.
 27. The method of claim 23,comprising modifying the T cells to express a T cell receptor havingantigenic specificity for a cancer antigen.